Sulfolobal phosphotriesterase-like (pll) lactonases activity having enhanced properties and the uses thereof

ABSTRACT

Mutated hyperthermophilic PTE having a lactonase activity derived from a hyperthermophilic phosphotriesterase corresponding to the consensus sequence of SEQ ID NO: 1, the mutated PTE including the at least one mutation chosen amongst 53 putative positions and the mutated PTE having enhanced properties. Also provided are compositions including the mutated hyperthermophilic PTE and the uses thereof, notably as bioscavenger of organophosphate compounds or as quorum quencher of the bacteria using lactones to communicate.

FIELD OF THE INVENTION

The present invention relates to Sulfolobal Phosphotriesterase-LikeLactonases (PLL) activity having enhanced properties and the usesthereof, notably as bioscavenger of organophosphorus compounds or asquorum quencher of the bacteria using lactones to communicate.

BACKGROUND OF THE INVENTION

Organophosphate (OPs) insecticides have become the most widely usedinsecticides available today. OPs are used in agriculture, at home, ingardens, and in veterinary practice. Since most of these compoundsinhibit some esterase enzymes, exposure to OPs can lead to serioustoxicity by multiple routes. Irreversible inhibition ofacetylcholinesterase by OPs, a key enzyme of the mammalian nervoussystem, causes severe damage for all vertebrates. Loss of enzymefunction leads to accumulation of acetylcholine in differentcompartments of the body causing muscle contraction, paralysis andrespiratory depression. Increased pulmonary secretions with respiratoryfailure are the usual causes of death from organophosphate poisoning.

Some of OPs have also been developed by armies before the World War II.The discovery of OPs with improved toxicity and/or higher stability haslead to the development of chemical warfar agents (CWA) such as sarin,soman, tabun or VX. Moreover, OPs insecticides, being easily accessibleand not so less toxic as compared to CWA OPs, constitute an importantrisk for the population. Faced with these growing threats, thedevelopment of anti-dotes has never been more urgent.

OPs are efficiently absorbed by inhalation, ingestion, and skinpenetration because of the hydrophobicity of these molecules. Theoccurrence of poisoning depends on the absorption rate of the compound.Symptoms of acute OPs poisoning develop during or after exposure, withinminutes to hours, depending of the method of the contact. Exposure byinhalation results in the fastest appearance of toxic symptoms, followedby the gastrointestinal route and finally dermal route.

Protective suits and masks do not always offer an effective protectionagainst OPs. In patients poisoned by OPs contamination of skin, clothingor hair, decontamination must proceed with surgical soap or laundrydetergents. Treatment of highly contaminated persons results inadministering atropine or diazepam which antagonize the effects ofexcessive concentrations of acetylcholine at end-organs havingmuscarinic receptors. Unfortunately, atropine remains ineffectiveagainst nicotinic actions, specifically muscle weakness and respiratorydepression in case of severe poisoning. Pralidoxime, a cholinesterasereactivator, relieves the nicotinic as well as the muscarinic effects ofOPs poisoning when administering less than 48 hours after poisoning. Theuse of this compound remains uneffective against sarin which holds avery quickly effect once inhalated. Clearing airway and improving tissueoxygenation is also very helpful.

Although some progress in prophylaxia has been made with theabovementioned techniques, existing protection and the treatments forthese poisoning nevertheless remain unsatisfactory.

The first OPs-hydrolases have been identified in several bacteria in theearly 90's (Cheng et al., 1993, Appl. Environ. Microbiol., 59:3138-3140, Raveh et al., 1993, Biochem Pharmacol., 45: 2465-2474). Theseenzymes are able to catalyze the hydrolysis of phosphoester bounds inOPs. Unfortunately, due to their low stoichiometric binding capacity toOPs, huge quantity of enzymes is needed to cure the poisoningindividuals. This renders the use of these enzymes disproportionate andquite expensive.

Some other microbial enzymes generally called phosphotriesterases (PTEs)show preferences for organophosphorous compounds with P—O or P—S bonds.These enzymes are members of the aminohydrolase superfamily, enzymescatalyzing hydrolysis of a broad range of compounds with differentchemical properties (phosphoesters, esters, amides, etc). Their codinggenes, opd (organo phosphate degradation), were isolated in soilbacteria such as Pseudomonas diminuta, also called Brevundominasdiminuta (Munnecke et al., 1976, Appl. Environ. Microbiol., 32: 7-13),Flavobacterium sp. (Sethunathan et al., 1973, Can J Microbiol, 19:873-875) and Agrobacterium radiobacter (Home et al., 2003, FEMSMicrobiol Lett, 222: 1-8), and genes similar to opd were also identifiedin Archaea (Merone et al., 2005, Extremophiles, 9: 297-305). Thecatalytic properties of hyperthermophilic PTEs are extensively studiedbecause of their ability to hydrolyze pesticides and several nerveagents (Jackson et al., 2005, Biochem Biophys Acta, 1752: 56-64/Jacksonet al., 2008, J Mol Biol, 375: 1189-1196/Wong et al., 2007,Biochemistry, 46: 13352-13369/Elias et al., 2008, J Mol Biol, 379:1017-1028/Pompea et al., 2009, Extremophiles, 13: 461-470). Thehyperthermophilic PTEs have the advantage of being very stable andinexpensive to produce due to their capacity to resist to organicsolvents or detergents at moderate temperature. Thus, hyperthermophilicPTEs are promising for the development of a bioscavenger for neurotoxicagents such as OPs.

Recently, three hyperthermophilic PTEs were isolated and purified fromSulfolobus sp.: SsoPox was isolated from Sulfolobus solfataricus (Meroneet al., 2005, Extremophiles, 9: 297-305), SacPox was isolated fromSulfolobus acidicaldarius (Porzio et al., 2007, Biochimie, 89: 625-636)and SisLac (also called SisPox) was isolated from Sulfolobus islandicus(Gotthard et al., 2011, Acta Crystallogr Sect F Struct Biol Cryst Commun67: 354-357/Hiblot et al., 2012, PLoS One 7: e47028). SsoPox, SacPox andSisLac are members of an enzyme family called phosphotriesterase-LikeLactonase (PLL). Phylogenetic and biochemical studies have revealed thatSsoPox and SisLac enzymes are native lactonases endowed with promiscuousparaoxonase activity and more generally with organophosphate hydrolaseactivity (Afriat et al., 2006, Biochemistry, 45: 13677-13686/Elias etal., 2012, J Biol Chem., 287(1): 11-20). Despite PTEs and PLLs enzymesexhibit the same (β/α)₈ barrel fold or so-called TIM barrel, theirability to hydrolyze different kinds of substrates such as lactones orOPs is different.

Lactones are signalling molecules synthesized by bacteria which allowthem to detect the population density. This cell-to-cell communicationprocess is termed quorum sensing (QS) and is well known to modulate manykey biological functions of bacteria including biofilm formation (Popatet al., 2008, British Medical Bulletin, 87: 63-75). This link between QSand virulence is central to the pathogenesis of many bacterialinfections, including P. aeruginosa (Sakuragi et al., 2007, J Bacteriol,189: 5383-5386) but also A. baumanii (Stacy et al., 2012, ACS Chem Biol,7(10): 1719-1728), Bulkolderia sp. (McKeon et al., 2011, J Infect Dis,February 1; 203(3):383-92), Vibrio sp. (Augustine et al., 2010, ArchMicrobiol 192(12): 1019-1022) or E. caratovora (Dong et al., 2001,Nature, 411: 813-817). Interfering with QS system, also called quorumquenching, is a promising approach to control bacterial diseases inplants and animals (Dong et al., 2001, nature, 411: 813-817).N-acylhomoserine lactones (AHLs) are molecules that mediate bacterialcommunication for many Gram negative bacteria and some Archaealorganisms (Zhang et al., 2012, ISME J., July; 6(7):1336-44). Itclassically regulates infection and virulence functions. These moleculesaccumulate in the media to reach a certain threshold for which thetranscriptional profile of the bacteria is altered (Hentzer et al.,2003, Embo J, 22: 3803-3815). By hydrolyzing AHLs, lactonases like PLLscan quench the AHL-mediated communication between bacteria, as seen forhuman paraoxonases (Ma et al., 2009, Appl Microbiol Biotechnol, 83:135-141) or AiiA lactonase (Dong et al., 2001, Nature, 411: 813-817).Because of their dual catalytic activities, lactonases andphosphotriesterases, PLLs constitute highly attractive candidate forbiotechnological utilization as quorum quenching agent or OPsbioscavenger.

In WO 2008/145865, the inventors of the present invention provide novelPTEs being more active vis-à-vis the OPs by introducing mutations inclose vicinity of the active site of SsoPox. The main aim of this workwas to obtain new enzymes with catalytic performance close to the onesof mesophilic PTEs.

SUMMARY OF THE INVENTION

Surprisingly, the inventors discovered that the introduction ofmutations in several β sheets or loops of the PLLs enzymes couldincrease not only the OPs hydrolyse activity but also the lactonasesactivity of said enzymes.

One aspect of the present invention is to provide, novel mutatedhyperthermophilic PTEs having a lactonase activity, having theadvantages of being both:

-   -   more active vis-à-vis the OPs, or more active vis-à-vis the        AHLs, or more active vis-à-vis the OPs and vis-à-vis the AHLs        than the wild type hyperthermophilic PTEs,    -   more stable and less expensive to produce than the mesophilic        PTEs.

Another aspect of the present invention contemplates a method for theestablishment of a library of mutated hyperthermophilic PTE variants.

Another aspect of the present invention is to provide efficient toolsfor the decontamination of OPs polluted surfaces of materials, of theskin, of hairs or mucous membranes. Said tools can be compositions,bioscavengers, cartridge decontamination, kit of decontamination,impregnated materials with new mutated hyperthermophilic PTEs.

Another aspect of the present invention is to provide vectors and hostcells able to synthesize the new mutated hyperthermophilic PTEs in largescale with a reduced cost.

Yet another aspect of the present invention is directed to the use ofnew mutated hyperthermophilic PTEs as bioscavengers within the contextof the decontamination of the surfaces of materials, of the skin ormucous membranes contaminated with organophosphorus compounds, or withinthe context of the pollution control of water polluted withorganophosphorus compounds, or within the context of the destruction ofstocks of neurotoxic agents.

Still another aspect of the present invention is to provide compositionscomprising new mutated hyperthermophilic PTEs for their use in thetreatment of diseases caused by bacteria using AHLs to communicate. Theexpression bacteria relates not only to bacteria but also to Archae.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1(A)-1(F): Chemical structures of SsoPox substrates

FIG. 1(A): The chemical structure of paraoxon

FIG. 1(B): The chemical structure of CMP-coumarin

FIG. 1(C): The chemical structure of 3-oxo-C12 AHL

FIG. 1(D): The chemical structure of 3-oxo-C10 AHL

FIG. 1(E): The chemical structure of undecanoic-δ-lactone

FIG. 1(F): The chemical structure of undecanoic-γ-lactone

FIGS. 2(A)-2(D): SsoPox phosphotriesterase activity screening andcharacterization

FIG. 2(A): Relative phosphotriesterase activities of W263 saturationsite variants have been screened with 1 mM of paraoxon substrate

FIG. 2(B): Relative phosphotriesterase activities of W263 saturationsite variants have been screened with 100 μM of paraoxon substrate.

FIG. 2(C): Relative phosphotriesterase activities of W263 saturationsite variants have been screened with 50 μM of CMP-coumarin substrate.

FIG. 2(D): Best variants (i.e. SsoPox W263F, W263M, W263A and W263L)have been characterized for paraoxon hydrolysis and catalyticefficiencies have been compared to SsoPox wt.

FIGS. 3(A)-3(C): SsoPox lactonase activity screening andcharacterization

FIG. 3(A): Schematic representation of P. aeruginosa based lactonaseactivity screnning method.

FIG. 3(B): Relative lactonase activity of W263 saturation sites variantshave been screened for 3-oxo-C12 AHL hydrolysis.

FIG. 3(C): Best variants (i.e. SsoPox W263I, W263V, W263T and W263A)have been characterized for 3-oxo-C12 AHL hydrolysis and catalyticefficiencies have been compared to SsoPox wt.

FIG. 4: SsoPox W263I mediated inhibition of lasB transcription.

The chart shows expression in treated cultures expressed as thepercentage of lasB expression in untreated control (no SsoPox W263I),and represents data averaged from three independent experiments, eachwith three technical replicates; error bars represent 95% confidenceintervals. Student's T test p=<0.05 for SsoPoxW263I. All T tests forcomparison of baseline with highest dose of enzyme.

FIG. 5: Inhibition of PAO1 biofilm formation by SsoPoxW263I.

Biofilms were grown in an MBEC device as described in the methodssection. Inhibition of P. aeruginosa biofilm formation by SsoPox W263Iis seen in a dose-dependent fashion: Student's T test p=<0.05 forSsoPoxW263I.

FIG. 6. Forty-eight hour survival curves of the 2 groups of animalsafter infection.

Animals were infected with 10⁸ CFU/mL (300 VaL) of P. aeruginosa PAO1and non-treated (NT) or immediately-treated (IT) with SsoPoxW263I.

FIGS. 7(A)-7(C): Lung histological examination after infection FIG.7(A): Pathological mapping of lungs representative of non-treated (NT)group: photomicrographs of pathological Giemsa staining X 100 of thelung sections. Mean histological severity score (HSS) was of (mean±SD)2,64±0.4 for the NT group.

FIG. 7(B): Pathological mapping of lungs representative ofdiffered-treatment (DT) group: photomicrographs of pathological Giemsastaining X 100 of the lung sections. Mean histological severity score(HSS) was of (mean±SD) 2.32±0.4 for the DT group (p=NS vs. NT).

FIG. 7(C): Pathological mapping of lungs representative ofimmediate-treatment (IT) group: photomicrographs of pathological Giemsastaining X 100 of the lung sections. Mean histological severity score(HSS) was of (mean±SD) 1.27±0.6 for the IT group (p=0.005 vs. NT).

DETAILED DESCRIPTION OF THE INVENTION

A subject of the invention is mutated hyperthermophilic PTE having alactonase activity derived from a hyperthermophilic phosphotriesterasecorresponding to the consensus sequence of SEQ ID NO: 1, said mutatedPTE comprise the at least one mutation selected from the groupconsisting of: substitutions of the glycine G in position 9, the lysineK in position 10, the valine V in position 29, the phenylalanine F orleucine L in position 48, the lysine K in position 56, the proline P inposition 69, the threonine T in position 70, the leucine L in position74, the isoleucine I in position 78, the valine V in position 85, thetyrosine Y in position 99, the tyrosine Y in position 101, theisoleucine I in position 124, the leucine L or serine S or asparagine Nin position 132, the aspartic acid D in position 143, the lysine K orasparagine N in position 166, the isoleucine I in position 169, theaspartic acid D in position 193, the glycine G in position 195, thearginine R in position 225, the glycine G in position 227, the leucine Lin position 228, the leucine L in position 230, the phenylalanine F inposition 231, the leucine L in position 232, the tyrosine Y position259, the cysteine C in position 260, the cysteine C in position 261, thethreonine T in position 262, the isoleucine I in position 263, theaspartic acid D in position 264, the tryptophane W in position 265, theglycine G in position 266, the threonine T or isoleucine I in position267, the alanine A in position 268, the lysine K or arginine R inposition 269, the proline P in position 270, the glutamic acid E inposition 271, the tyrosine Y or leucine L in position 272, the lysine Kin position 273, the proline P in position 274, the lysine K in position275, the leucine L in position 276, the alanine A in position 277, theproline P in position 278, the arginine R or lysine K in position 279,the tryptophan W in position 280, the serine S in position 281, theisoleucine I or methionine M in position 282, the threonine T or alanineA or serine S in position 283, the leucine L in position 284, theisoleucine I in position 285, the asparagine N or serine S or threonineT in position 299, of SEQ ID NO: 1 by any other natural amino aciddifferent from the one(s) described in the consensus sequence, with anexception for positions 48, 132, 166, 267, 269, 272, 279, 282, 283 and299 where the substitution can be done with one amino acid described inthe consensus sequence only if said substitution on said positions isalways associated with at least another substitution chosen among theabove-mentioned positions, or by any other non-natural amino acid, withthe proviso that when the at least one mutation is selected from thegroup consisting of substitutions of the tyrosine Y in position 99, thetyrosine Y in position 101, the arginine R in position 225, the cysteineC in position 260, then the said at least one mutation is alwaysassociated with at least one mutation selected from the group consistingof substitutions of the glycine G in position 9, the lysine K inposition 10, the phenylalanine F or leucine L in position 48, the lysineK in position 56, the isoleucine I in position 78, the valine V inposition 85, the isoleucine I in position 124, the leucine L or serine Sor asparagine N in position 132, the lysine K or asparagine N inposition 166, the isoleucine I in position 169, the aspartic acid D inposition 193, the glycine G in position 195, the leucine L in position230, the leucine L in position 232, the tyrosine Y in position 259, thecysteine C in position 261, the threonine T in position 262, theisoleucine I in position 263, the aspartic acid D in position 264, theglycine G in position 266, the threonine T or isoleucine I in position267, the alanine A in position 268, the lysine K or arginine R inposition 269, the proline P in position 270, the glutamic acid E inposition 271, the tyrosine Y or leucine L in position 272, the lysine Kin position 273, the proline P in position 274, the lysine K in position275, the leucine L in position 276, the alanine A in position 277, theproline P in position 278, the arginine R or lysine K in position 279,the serine S in position 281, the isoleucine I or methionine M inposition 282, the threonine T or alanine A or serine S in position 283,the leucine L in position 284, the isoleucine I in position 285, theasparagine N or serine S or threonine T in position 299,

and with the proviso that when the at least one mutation selected fromthe group consisting of substitutions of the valine V in position 29,the proline P in position 69, the threonine T in position 70, theleucine L in position 74, the aspartic acid D in position 143, theglycine G in position 227, the leucine L in position 228, thephenylalanine F in position 231, the tryptophane W in position 265, thetryptophane W in position 280 is associated with the at least onemutation selected from the group consisting of the substitutions of thetyrosine Y in position 99, the tyrosine Y in position 101, the arginineR in position 225, the cysteine C in position 260 to form associatedmutations, then the said associated mutations are always associated withat least one mutation selected from the group consisting ofsubstitutions of the glycine G in position 9, the lysine K in position10, the phenylalanine F or leucine L in position 48, the lysine K inposition 56, the isoleucine I in position 78, the valine V in position85, the isoleucine I in position 124, the leucine L or serine S orasparagine N in position 132, the lysine K or asparagine N in position166, the isoleucine I in position 169, the aspartic acid D in position193, the glycine G in position 195, the leucine L in position 230, theleucine L in position 232, the tyrosine Y in position 259, the cysteineC in position 261, the threonine T in position 262, the isoleucine I inposition 263, the aspartic acid D in position 264, the glycine G inposition 266, the threonine T or isoleucine I in position 267, thealanine A in position 268, the lysine K or arginine R in position 269,the proline P in position 270, the glutamic acid E in position 271, thetyrosine Y or leucine L in position 272, the lysine K in position 273,the proline P in position 274, the lysine K in position 275, the leucineL in position 276, the alanine A in position 277, the proline P inposition 278, the arginine R or lysine K in position 279, the serine Sin position 281, the isoleucine I or methionine M in position 282, thethreonine T or alanine A or serine S in position 283, the leucine L inposition 284, the isoleucine I in position 285, the asparagine N orserine S or threonine T in position 299.

PTEs are zinc-metalloproteases that were originally identified for theirability to hydrolyse phosphotriesterase-containing organophosphorouscompounds, but recently more members of this family were found topossess lactonase activity as well. Lactonase activity is the ability tohydrolyze the ester bound in the lactone ring.

The expression “mutated hyperthermophilic PTE having a lactonaseactivity” relates to any enzyme having both lactonase andphosphotriesterase catalytic activities, said enzymes being isolatedfrom thermophilic or hyperthermophilic bacteria belonging to the PLLs orPTEs superfamilies. By “superfamiliy” is meant a large group of proteinssharing the same fold (topology and secondary structure elements), andthe same active site architecture. A superfamily is comprised of dozensof groups of proteins sharing the same three dimensional structure andfunctions, each group exhibiting a different function. These functionstypically share a common element (e.g. a key chemical step in enzymecatalysis) and also the active site residues executing this element. By“thermophilic bacteria” are meant bacteria living between 45° C. to 120°C. By “hyperthermophilic bacteria” is meant bacteria for which theoptimal temperatures are above 80° C. The thermostability of the enzymesisolated from thermophilic or hyperthermophilic bacteria confers themthe advantage of being inexpensive to produce, on the one hand becausethey are stable in organic solvents which make them more suitable forindustrial processes, and, on the other hand, because they are veryinexpensive to purify by the technique of heating the cell lysates ofthe cells producing the above-mentioned enzymes; a large yield and highpurity are thus obtained in one stage.

Lactonase and phosphotriesterase catalytic activities can be tested ontheir respective substrates according to methods disclosed inexperimental part of the invention.

The introduction of an amino acid residue in position 2 of SEQ ID NO: 1results from the experimental protocols used to perform the differentmutated hyperthermophilic PTEs, notably due to the choice of restrictionenzyme in the cloning site of vectors for the building of the mutatedhyperthermophilic PTEs. For example, the use of NcoI restriction enzymein the cloning site of said vectors leads to the addition of the alanineresidue in position 2 of SEQ ID NO: 1 in order to avoid a change in thereading frame. The introduction of said alanine residue in position 2 ofSEQ ID NO: 1 has no effect in the activity of either the wild type orthe mutated hyperthermophilic PTEs. It means that two mutatedhyperthermophilic PTEs having a sequence derived from SEQ ID NO: 1, onebearing an added alanine residue in position 2, the other one being freeof said alanine residue in position 2 share exactly the same enzymaticactivity in terms of performance.

For positions 48, 132, 166, 267, 269, 272, 279, 282, 283 and 299, thesubstitution can be done with one of the amino acid described in theconsensus sequence, i.e. already existing in natural hyperthermophilicPTEs only if said substitution on said positions is always associatedwith at least any other substitution chosen among the above-mentionedposition. For example, if phenylalanine F in position 48 is substitutedby a leucine L, then another substitution should be done at least in anyof the 52 other positions as disclosed.

The first proviso aims to exclude a single mutation at positions Y99,Y101, R225 or C260 of SEQ ID NO: 1. When the natural amino acid at oneof the above-mentioned position is mutated, then it is always associatedwith at least one the 39 substitutions in position G9, K10, F/L48, K56,I78, V85, I124, L/S/N132, K/N166, I169, D193, G195, L230, L232, Y259,C261, T262, 1263, D264, G266, T/I267, A268, K/R269, P270, E271, Y/L272,K273, P274, K275, L276, A277, P278, R/K279, S281, I/M282, T/A/S283,L284, 1285, N/S/T299 of SEQ ID NO: 1.

The second proviso aims to exclude all the combinations of at least onemutation selected from the group consisting of substitution of thevaline V in position 29, substitution of the proline P in position 69,substitution of the threonine T in position 70, substitution of theleucine L in position 74, substitution of the aspartic acid D inposition 143, substitution of the glycine G in position 227,substitution of the leucine L in position 228, substitution of thephenylalanine F in position 231, substitution of the tryptophane W inposition 265, substitution of the tryptophane W in position 280associated with at least one mutation selected from the group consistingof the tyrosine Y in position 99, substitution of the tyrosine Y inposition 101, substitution of the arginine R in position 225,substitution of the cysteine C in position 260 of SEQ ID NO: 1. Whensuch a combination of mutations occurred, then it is always associatedwith at least one the 39 substitutions in position G9, K10, F/L48, K56,I78, V85, I124, L/S/N132, K/N166, I169, D193, G195, L230, L232, Y259,C261, T262, I263, D264, G266, T/I267, A268, K/R269, P270, E271, Y/L272,K273, P274, K275, L276, A277, P278, R/K279, S281, I/M282, T/A/S283,L284, 1285, N/S/T299 of SEQ ID NO: 1.

The aim of the above-mentioned proviso is to exclude some specificmutated hyperthermophilic phosphotriesterase (PTEs) previously disclosedby the inventor in WO 2008/145865.

The mutated hyperthermophilic phosphotriesterase (PTEs) having alactonase activity of the invention have the advantage of being moreactive than the wild type hyperthermophilic phosphotriesterase (PTEs)having a lactonase activity from which they derived not only within thecontext of hydrolysis of OPs but also within the context of thetreatment of diseases caused by bacteria using AHLs to communicate,notably by hydrolysis of AHLs.

The hyperthermophilic PTEs having a lactonase activity of the presentinvention also have the advantage of being more active:

-   -   within the context of the hydrolysis of the OPs, and/or,    -   within the context of quorum quenching, i.e. within the context        of resistance to pathogen infections, than the wild type        hyperthermophilic PTEs from which they derived.

In a preferred embodiment, the mutated hyperthermophilicphosphotriesterase (PTE) having a lactonase activity derived from ahyperthermophilic phosphotriesterase according to the present invention,wherein the amino acid in position 2 in SEQ ID NO: 1 is missing, saidmutated PTE comprise the at least one mutation selected from the groupconsisting of: substitutions of the glycine G in position 8, the lysineK in position 9, the valine V in position 28, the phenylalanine F orleucine L in position 47, the lysine K in position 55, the proline P inposition 68, the threonine T in position 69, the leucine L in position73, the isoleucine I in position 77, the valine V in position 84, thetyrosine Y in position 98, the tyrosine Y in position 100, theisoleucine I in position 123, the leucine L or serine S or asparagine Nin position 131, the aspartic acid D in position 142, the lysine K orasparagine N in position 165, the isoleucine I in position 168, theaspartic acid D in position 192, the glycine G in position 194, thearginine R in position 224, the glycine G in position 226, the leucine Lin position 227, the leucine L in position 229, the phenylalanine F inposition 230, the leucine L in position 231, the tyrosine Y position258, the cysteine C in position 259, the cysteine C in position 260, thethreonine T in position 261, the isoleucine I in position 262, theaspartic acid D in position 263, the tryptophane W in position 264, theglycine G in position 265, the threonine T or isoleucine I in position266, the alanine A in position 267, the lysine K or arginine R inposition 268, the proline P in position 269, the glutamic acid E inposition 270, the tyrosine Y or leucine L in position 271, the lysine Kin position 272, the proline P in position 273, the lysine K in position274, the leucine L in position 275, the alanine A in position 276, theproline P in position 277, the arginine R or lysine K in position 278,the tryptophan W in position 279, the serine S in position 280, theisoleucine I or methionine M in position 281, the threonine T or alanineA or serine S in position 282, the leucine L in position 283, theisoleucine I in position 284, the asparagine N or serine S or threonineT in position 298, of SEQ ID NO: 1 by any other natural amino aciddifferent from the one(s) described in the consensus sequence,

with an exception for positions 47, 131, 165, 266, 268, 271, 278, 281,282 and 298 where the substitution can be done with one amino aciddescribed in the consensus sequence only if said substitution on saidpositions is always associated with at least another substitution chosenamong the above-mentioned positions, or by any other non-natural aminoacid, with the proviso that when the at least one mutation is selectedfrom the group consisting of substitutions of the tyrosine Y in position98, the tyrosine Y in position 100, the arginine R in position 224, thecysteine C in position 259, then the said at least one mutation isalways associated with at least one mutation selected from the groupconsisting of substitutions of the glycine G in position 8, the lysine Kin position 9, the phenylalanine F or leucine L in position 47, thelysine K in position 55, the isoleucine I in position 77, the valine Vin position 84, the isoleucine I in position 123, the leucine L orserine S or asparagine N in position 131, the lysine K or asparagine Nin position 165, the isoleucine I in position 168, the aspartic acid Din position 192, the glycine G in position 194, the leucine L inposition 229, the leucine L in position 231, the tyrosine Y in position258, the cysteine C in position 260, the threonine T in position 261,the isoleucine I in position 262, the aspartic acid D in position 263,the glycine G in position 265, the threonine T or isoleucine I inposition 266, the alanine A in position 267, the lysine K or arginine Rin position 268, the proline P in position 269, the glutamic acid E inposition 270, the tyrosine Y or leucine L in position 271, the lysine Kin position 272, the proline P in position 273, the lysine K in position274, the leucine L in position 275, the alanine A in position 276, theproline P in position 277, the arginine R or lysine K in position 278,the serine S in position 280, the isoleucine I or methionine M inposition 281, the threonine T or alanine A or serine S in position 282,the leucine L in position 283, the isoleucine I in position 284, theasparagine N or serine S or threonine T in position 298,and with the proviso that when the at least one mutation selected fromthe group consisting of substitutions of the valine V in position 28,the proline P in position 68, the threonine T in position 69, theleucine L in position 73, the aspartic acid D in position 142, theglycine G in position 226, the leucine L in position 227, thephenylalanine F in position 230, the tryptophane W in position 264, thetryptophane W in position 279 is associated with the at least onemutation selected from the group consisting of the substitutions of thetyrosine Y in position 98, the tyrosine Y in position 100, the arginineR in position 224, the cysteine C in position 259 to form associatedmutations, then the said associated mutations are always associated withat least one mutation selected from the group consisting ofsubstitutions of the glycine G in position 8, the lysine K in position9, the phenylalanine F or leucine L in position 47, the lysine K inposition 55, the isoleucine I in position 77, the valine V in position84, the isoleucine I in position 123, the leucine L or serine S orasparagine N in position 131, the lysine K or asparagine N in position165, the isoleucine I in position 168, the aspartic acid D in position192, the glycine G in position 194, the leucine L in position 229, theleucine L in position 231, the tyrosine Y in position 258, the cysteineC in position 260, the threonine T in position 261, the isoleucine I inposition 262, the aspartic acid D in position 263, the glycine G inposition 265, the threonine T or isoleucine I in position 266, thealanine A in position 267, the lysine K or arginine R in position 268,the proline P in position 269, the glutamic acid E in position 270, thetyrosine Y or leucine L in position 271, the lysine K in position 272,the proline P in position 273, the lysine K in position 274, the leucineL in position 275, the alanine A in position 276, the proline P inposition 277, the arginine R or lysine K in position 278, the serine Sin position 280, the isoleucine I or methionine M in position 281, thethreonine T or alanine A or serine S in position 282, the leucine L inposition 283, the isoleucine I in position 284, the asparagine N orserine S or threonine T in position 298.

In this preferred embodiment, the alanine residue in position 2 isabsent of the SEQ ID NO: 1.

The first proviso aims to exclude a single mutation at positions Y98,Y100, R224 or C259 of SEQ ID NO: 1. When the natural amino acid at oneof the above-mentioned position is mutated, then it is always associatedwith at least one the 39 substitutions in position G8, K9, F/L47, K55,I77, V84, I123, L/S/N131, K/N165, I168, D192, G194, L229, L231, Y258,C260, T261, 1262, D263, G265, T/I266, A267, K/R268, P269, E270, Y/L271,K272, P273, K274, L275, A276, P277, R/K278, S280, I/M281, T/A/S282,L283, 1284, N/S/T298 of SEQ ID NO: 1.

The second proviso aims to exclude all the combinations of at least onemutation selected from the group consisting of substitution of thevaline V in position 28, substitution of the proline P in position 68,substitution of the threonine T in position 69, substitution of theleucine L in position 73, substitution of the aspartic acid D inposition 142, substitution of the glycine G in position 226,substitution of the leucine L in position 227, substitution of thephenylalanine F in position 230, substitution of the tryptophane W inposition 264, substitution of the tryptophane W in position 279associated with at least one mutation selected from the group consistingof the tyrosine Y in position 98, substitution of the tyrosine Y inposition 100, substitution of the arginine R in position 224,substitution of the cysteine C in position 259 of SEQ ID NO: 1. Whensuch a combination of mutations occurred, then it is always associatedwith at least one the 39 substitutions in position G8, K9, F/L47, K55,I77, V84, I123, L/S/N131, K/N165, 1168, D192, G194, L229, L231, Y258,C260, T261, I262, D263, G265, T/I266, A267, K/R268, P269, E270, Y/L271,K272, P273, K274, L275, A276, P277, R/K278, S280, I/M281, T/A/S282,L283, 1284, N/S/T298 of SEQ ID NO: 1.

In a more preferred embodiment, the mutated hyperthermophilic PTEshaving a lactonase activity according to the present inventioncorresponding to the sequence of SEQ ID NO: 3 or having at least 70% ormore identity to the amino acid sequence of SEQ ID NO: 3, said mutatedPTE comprise the at least one mutation selected from the groupconsisting of: substitutions of the glycine G in position 7, the lysineK in position 8, the valine V in position 27, the phenylalanine F inposition 46, the lysine K in position 54, the proline P in position 67,the threonine T in position 68, the leucine L in position 72, theisoleucine I in position 76, the valine V in position 83, the tyrosine Yin position 97, the tyrosine Y in position 99, the isoleucine I inposition 122, the leucine L in position 130, the aspartic acid D inposition 141, the lysine K in position 164, the isoleucine I in position167, the aspartic acid D in position 191, the glycine G in position 193,the arginine R in position 223, the glycine G in position 225, theleucine L in position 226, the leucine L in position 228, thephenylalanine F in position 229, the leucine L in position 230, thetyrosine Y position 257, the cysteine C in position 258, the cysteine Cin position 259, the threonine T in position 260, the isoleucine I inposition 261, the aspartic acid D in position 262, the tryptophane W inposition 263, the glycine G in position 264, the threonine T in position265, the alanine A in position 266, the lysine K in position 267, theproline P in position 268, the glutamic acid E in position 269, thetyrosine Y in position 270, the lysine K in position 271, the proline Pin position 272, the lysine K in position 273, the leucine L in position274, the alanine A in position 275, the proline P in position 276, thearginine R in position 277, the tryptophan W in position 278, the serineS in position 279, the isoleucine I in position 280, the threonine T inposition 281, the leucine L in position 282, the isoleucine I inposition 283, the asparagine N in position 297, of SEQ ID NO: 3 by anyother natural or non-natural amino acid,

with the proviso that when the at least one mutation is selected fromthe group consisting of substitutions of the tyrosine Y in position 97,the tyrosine Y in position 99, the arginine R in position 223, thecysteine C in position 258, then the said at least one mutation isalways associated with at least one mutation selected from the groupconsisting of substitutions of the glycine G in position 7, the lysine Kin position 8, the phenylalanine F in position 46, the lysine K inposition 54, the isoleucine I in position 76, the valine V in position83, the isoleucine I in position 122, the leucine L in position 130, thelysine K in position 164, the isoleucine I in position 167, the asparticacid D in position 191, the glycine G in position 193, the leucine L inposition 228, the leucine L in position 230, the tyrosine Y in position257, the cysteine C in position 259, the threonine T in position 260,the isoleucine I in position 261, the aspartic acid D in position 262,the glycine G in position 264, the threonine T in position 265, thealanine A in position 266, the lysine K in position 267, the proline Pin position 268, the glutamic acid E in position 269, the tyrosine Y inposition 270, the lysine K in position 271, the proline P in position272, the lysine K in position 273, the leucine L in position 274, thealanine A in position 275, the proline P in position 276, the arginine Rin position 277, the serine S in position 279, the isoleucine I inposition 280, the threonine T in position 281, the leucine L in position282, the isoleucine I in position 283, the asparagine N in position 297,and with the proviso that when the at least one mutation selected fromthe group consisting of substitutions of the valine V in position 27,the proline P in position 67, the threonine T in position 68, theleucine L in position 72, the aspartic acid D in position 141, theglycine G in position 225, the leucine L in position 226, thephenylalanine F in position 229, the tryptophane W in position 263, thetryptophane W in position 278 is associated with the at least onemutation selected from the group consisting of the substitutions of thetyrosine Y in position 97, the tyrosine Y in position 99, the arginine Rin position 223, the cysteine C in position 258 to form associatedmutations, then the said associated mutations are always associated withat least one mutation selected from the group consisting ofsubstitutions of the glycine G in position 7, the lysine K in position8, the phenylalanine F in position 46, the lysine K in position 54, theisoleucine I in position 76, the valine V in position 83, the isoleucineI in position 122, the leucine L in position 130, the lysine K inposition 164, the isoleucine I in position 167, the aspartic acid D inposition 191, the glycine G in position 193, the leucine L in position228, the leucine L in position 230, the tyrosine Y in position 257, thecysteine C in position 259, the threonine T in position 260, theisoleucine I in position 261, the aspartic acid D in position 262, theglycine G in position 264, the threonine T in position 265, the alanineA in position 266, the lysine K in position 267, the proline P inposition 268, the glutamic acid E in position 269, the tyrosine Y inposition 270, the lysine K in position 271, the proline P in position272, the lysine K in position 273, the leucine L in position 274, thealanine A in position 275, the proline P in position 276, the arginine Rin position 277, the serine S in position 279, the isoleucine I inposition 280, the threonine T in position 281, the leucine L in position282, the isoleucine I in position 283, the asparagine N in position 297.

In this more preferred embodiment, the alanine residue in position 2 andthe threonine residue in position 3 are absent of the SEQ ID NO: 1.

The first proviso aims to exclude a single mutation at positions Y97,Y99, R223 or C258 of SEQ ID NO: 1. When the natural amino acid at one ofthe above-mentioned position is mutated, then it is always associatedwith at least one the 39 substitutions in position G7, K8, F46, K54,I76, V83, I122, L130, K164, 1167, D191, G193, L228, L230, Y257, C259,T260, I261, D262, G264, T265, A266, K267, P268, E269, Y270, K271, P272,K273, L274, A275, P276, R277, S279, 1280, T281, L282, 1283, N297 of SEQID NO: 1.

The second proviso aims to exclude all the combinations of at least onemutation selected from the group consisting of substitution of thevaline V in position 27, substitution of the proline P in position 67,substitution of the threonine T in position 68, substitution of theleucine L in position 72, substitution of the aspartic acid D inposition 141, substitution of the glycine G in position 225,substitution of the leucine L in position 226, substitution of thephenylalanine F in position 229, substitution of the tryptophane W inposition 263, substitution of the tryptophane W in position 278associated with at least one mutation selected from the group consistingof the tyrosine Y in position 97, substitution of the tyrosine Y inposition 99, substitution of the arginine R in position 223,substitution of the cysteine C in position 258 of SEQ ID NO: 1. Whensuch a combination of mutations occurred, then it is always associatedwith at least one the 39 substitutions in position G7, K8, F46, K54,I76, V83, I122, L130, K164, I167, D191, G193, L228, L230, Y257, C259,T260, 1261, D262, G264, T265, A266, K267, P268, E269, Y270, K271, P272,K273, L274, A275, P276, R277, S279, 1280, T281, L282, 1283, N297 of SEQID NO: 1.

The invention relates more particularly to the above-mentioned mutatedhyperthermophilic PTEs having a lactonase activity, derived from ahyperthermophilic phosphotriesterase according to the present invention,wherein the amino acid in position 2 in SEQ ID NO: 1 is missing, saidmutated PTEs comprise the at least one mutation selected from the groupconsisting of: substitutions of the valine V in position 28, the prolineP in position 68, the threonine T in position 69, the leucine L inposition 73, the tyrosine Y in position 98, the tyrosine Y in position100, the aspartic acid D in position 142, the arginine R in position224, the glycine G in position 226, the leucine L in position 227, thephenylalanine F in position 230, the cysteine C in position 259, thetryptophane W in position 264 and the tryptophan W in position 279, ofSEQ ID NO: 1 by any other natural amino acid different from the one(s)described in the consensus sequence or by any other non-natural aminoacid.

In an even more specific embodiment, the above-mentioned mutatedhyperthermophilic PTEs having a lactonase activity, derived from ahyperthermophilic phosphotriesterase according to the present invention,wherein the amino acid in position 2 in SEQ ID NO: 1 is missing,comprise only one mutation, said mutation being selected from the groupconsisting of: substitutions of the valine V in position 28, the prolineP in position 68, the threonine T in position 69, the leucine L inposition 73, the aspartic acid D in position 142, the glycine G inposition 226, the leucine L in position 227, the phenylalanine F inposition 230, the tryptophane W in position 264 and the tryptophan W inposition 279, of SEQ ID NO: 1 by any other natural amino acid differentfrom the one(s) described in the consensus sequence or by any othernon-natural amino acid.

In a more specific embodiment, the above-mentioned mutatedhyperthermophilic PTEs having a lactonase activity, derived from ahyperthermophilic phosphotriesterase according to the present invention,wherein the amino acid in position 2 in SEQ ID NO: 1 is missing,comprise the at least one mutation selected from the group consistingof: substitutions of the glycine G in position 8, the lysine K inposition 9, the phenylalanine F or leucine L in position 47, the lysineK in position 55, the isoleucine I in position 77, the valine V inposition 84, the isoleucine I in position 123, the leucine L or serine Sor asparagine N in position 131, the lysine K or asparagine N inposition 165, the isoleucine I in position 168, the aspartic acid D inposition 192, the glycine G in position 194, the leucine L in position229, the leucine L in position 231, the tyrosine Y position 258, thecysteine C in position 260, the threonine T in position 261, theisoleucine I in position 262, the aspartic acid D in position 263, theglycine G in position 265, the threonine T or isoleucine I in position266, the alanine A in position 267, the lysine K or arginine R inposition 268, the proline P in position 269, the glutamic acid E inposition 270, the tyrosine Y or leucine L in position 271, the lysine Kin position 272, the proline P in position 273, the lysine K in position274, the leucine L in position 275, the alanine A in position 276, theproline P in position 277, the arginine R or lysine K in position 278,the serine S in position 280, the isoleucine I or methionine M inposition 281, the threonine T or alanine A or serine S in position 282,the leucine L in position 283, the isoleucine I in position 284 and theasparagine N or serine S or threonine T in position 298, of SEQ ID NO: 1by any other natural amino acid different from the one(s) described inthe consensus sequence or by any other non-natural amino acid.

In an even more specific embodiment, the above-mentioned mutatedhyperthermophilic PTEs having a lactonase activity, derived from ahyperthermophilic phosphotriesterase according to the present invention,wherein the amino acid in position 2 in SEQ ID NO: 1 is missing,comprise only one mutation, said mutation being selected from the groupconsisting of: substitutions of the glycine G in position 8, the lysineK in position 9, the phenylalanine F or leucine L in position 47, thelysine K in position 55, the isoleucine I in position 77, the valine Vin position 84, the isoleucine I in position 123, the leucine L orserine S or asparagine N in position 131, the lysine K or asparagine Nin position 165, the isoleucine I in position 168, the aspartic acid Din position 192, the glycine G in position 194, the leucine L inposition 229, the leucine L in position 231, the tyrosine Y position258, the cysteine C in position 260, the threonine T in position 261,the isoleucine I in position 262, the aspartic acid D in position 263,the glycine G in position 265, the threonine T or isoleucine I inposition 266, the alanine A in position 267, the lysine K or arginine Rin position 268, the proline P in position 269, the glutamic acid E inposition 270, the tyrosine Y or leucine L in position 271, the lysine Kin position 272, the proline P in position 273, the lysine K in position274, the leucine L in position 275, the alanine A in position 276, theproline P in position 277, the arginine R or lysine K in position 278,the serine S in position 280, the isoleucine I or methionine M inposition 281, the threonine T or alanine A or serine S in position 282,the leucine L in position 283, the isoleucine I in position 284 and theasparagine N or serine S or threonine T in position 298, of SEQ ID NO: 1by any other natural amino acid different from the one(s) described inthe consensus sequence or by any other non-natural amino acid.

A more particular subject of the invention is the above-mentionedmutated hyperthermophilic PTEs having a lactonase activity, derived fromthe hyperthermophilic PTE of Sulfolobus solfataricus corresponding tothe sequence SEQ ID NO: 3, or from the hyperthermophilic PTE ofSulfolobus acidocaldarius corresponding to the sequence SEQ ID NO: 5, orfrom the hyperthermophilic PTE of Sulfolobus islandicus corresponding tothe sequence SEQ ID NO: 7, said sequences SEQ ID NO: 3, SEQ ID NO: 5 andSEQ ID NO: 7 belonging to the consensus sequence SEQ ID NO: 1, the aminoacids in position 2 in SEQ ID NO: 1 being missing from SEQ ID NO: 5 andthe amino acids in position 2 and 3 in SEQ ID NO: 1 being missing fromSEQ ID NO: 3 and SEQ ID NO: 7.

The invention relates more particularly to the above-mentioned mutatedhyperthermophilic PTEs having a lactonase activity, derived from ahyperthermophilic phosphotriesterase according to the present invention,wherein the amino acid in position 2 in SEQ ID NO: 1 is missing, andwherein the at least one mutation is selected from the group consistingof:

-   -   substitution of the glycine G in position 8 by a non-bulky amino        acid selected from the group consisting of PLIVADCSTN, in        particular STA, notably S,    -   substitution of the lysine K in position 9 by a charged amino        acid selected from the group consisting of RHDEC, in particular        EDR, notably E,    -   substitution of the valine V in position 28 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular GIFA, notably A,    -   substitution of the phenylalanine F in position 47 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN or by a polar amino acid selected from the group        consisting of WYSTCQNRKHDE, in particular LYW, notably L, or        substitution of the leucine L in position 47 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular YW,    -   substitution of the lysine K in position 55 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular IRL, notably I,    -   substitution of the threonine T in position 69 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSN, in        particular VAS, notably S,    -   substitution of the leucine L in position 73 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular CAMI, notably I,    -   substitution of the isoleucine I in position 77 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular PTV, notably T,    -   substitution of the valine V in position 84 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular AGI, notably A,    -   substitution of the tyrosine Y in position 98 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular FCLW, notably W,    -   substitution of the tyrosine Y in position 100 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular GEWF, notably F,    -   substitution of the isoleucine I in position 123 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular LAV, notably L,    -   substitution of the leucine L or serine S or asparagine N in        position 131 by a non-bulky amino acid selected from the group        consisting of GPIVADCT, in particular PT, notably P,    -   substitution of the aspartic acid D in position 142 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE, in        particular SET, notably T,    -   substitution of the lysine K in position 165 by a polar amino        acid selected from the group consisting of WYSTCNQRHDE, in        particular NQR, notably N, or substitution of the asparagine N        in position 165 by a polar amino acid selected from the group        consisting of WYSTCQRKHDE, in particular QR,    -   substitution of the glycine G in position 194 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCNQRKHDE, in particular ST, notably S,    -   substitution of the arginine R in position 224 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNKHDE, in particular CSTAH, notably AC,    -   substitution of the leucine L in position 227 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by apolar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular AIVH, notably V,    -   substitution of the leucine L in position 229 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VIMFGAPWYC, in particular IM, notably M,    -   substitution of the phenylalanine F in position 230 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN, in particular LTAS, notably S,    -   substitution of the leucine L in position 231 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN, in        particular PVA, notably P,    -   substitution of the cysteine C in position 259 by a hydrophobic        amino acid selected from the group consisting of VILMFGAPWY, in        particular YLIA, notably LA,    -   substitution of the isoleucine I in position 262 by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWYC, in particular FWC, notably F,    -   substitution of the tryptophane W in position 264 by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPYC or by a non-bulky amino acid selected from the group        consisting of GPLIVADCSTN, in particular ALMFCITV, notably F,        with the proviso that the tryptophane W in position 264 can not        be substituted by a phenylalanine F in SEQ ID NO: 3,    -   substitution of the leucine L in position 275 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular AVP, notably P,    -   substitution of the alanine A in position 276 by a hydrophobic        amino acid selected from the group consisting of VILMFGPWYC or        by a non-bulky amino acid selected from the group consisting of        GPLIVDCSTN, in particular NVMT, notably T,    -   substitution of the isoleucine I or methionine M in position 281        by a non-bulky amino acid selected from the group consisting of        GPLVADCSTN or by a hydrophobic amino acid selected from the        group consisting of VLFGAPWYC, in particular TYP, notably T,    -   substitution of the leucine L in position 283 by a bulky amino        acid selected from the group consisting of EKHRQYWFM, in        particular FMH, notably M,    -   and substitution of the asparagine N or serine S or threonine T        in position 298 by a polar amino acid selected from the group        consisting of WYCQRKHDE, notably Q.

These 29 particular substitutions in position G8, K9, V28, F/L47, K55,T69, L73, I77, V84, Y98, Y100, I123, L/S/N131, D142, K/N165, G194, R224,L227, L229, F230, L231, C259, 1262, W264, L275, A276, I/M281, L283 andN/S/T298 belong to the first set of substitutions called set 1.

These positions are considered as key positions to modulate enzymaticactivities and are also implicated in AHLs substrate accommodationwithin the active site of the enzyme. Said positions had been identifiedby directed evolution strategy.

By the term “substitution” is meant the replacement of one amino acid byanother. The substitutions can be conservative, i.e. the substitutedamino acid is replaced by an amino acid of the same structure or withthe same physico-chemical properties (polar, hydrophobic, acidic, basicamino acids) such that the three dimensional structure of the proteinremains unchanged, or by contrast non conservative.

When set 1 is related to a sequence, it means that at least onesubstitution of said set occurs in said sequence.

The invention relates more particularly to the above-mentioned mutatedhyperthermophilic PTE having a lactonase activity, derived from ahyperthermophilic phosphotriesterase according to the present invention,wherein the amino acid in position 2 in SEQ ID NO: 1 is missing, andwherein the at least one mutation is selected from the group consistingof:

-   -   substitution of the isoleucine I in position 168 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VLMFGAPWYC, in particular VAL, notably V,    -   substitution of the aspartic acid D in position 192 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN or by a polar amino acid selected from the group        consisting of WYSTCQNRKHE, in particular ST, notably S,    -   substitution of the tyrosine Y position 258 by a non-bulky amino        acid selected from the group consisting of GPLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWC, in particular CSVW, notably C,    -   substitution of the cysteine C in position 260 a non-bulky amino        acid selected from the group consisting of GPLIVADSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWY, in particular SFWV, notably S,    -   substitution of the threonine T in position 261 a non-bulky        amino acid selected from the group consisting of GPLIVADCSN or        by a polar amino acid selected from the group consisting of        WYSCQNRKHE, in particular GH, notably G,    -   substitution of the aspartic acid D in position 263 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE or        by a non-bulky amino acid selected from the group consisting of        GPLIVACSTN, in particular SLH,    -   substitution of the glycine G in position 265 non-bulky amino        acid selected from the group consisting of PLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFAPWYC, in particular AVP,    -   substitution of the threonine T or isoleucine I in position 266        by a hydrophobic amino acid selected from the group consisting        of VLMFGAPWYC or by a non-bulky amino acid selected from the        group consisting of GPLVADCSN, in particular VWP, notably V,    -   substitution of the alanine A in position 267 by a polar amino        acid selected from the group consisting of WYSTCQNRKHDE, in        particular NQ, notably N,    -   substitution of the lysine K or arginine R in position 268 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN or by a hydrophobic amino acid selected from the        group consisting of VILMFGAPWYC, in particular IAP, notably IP,    -   substitution of the proline P in position 269 by a hydrophobic        amino acid selected from the group consisting of VILMFGACWY, in        particular MCL, notably M,    -   substitution of the glutamic acid E in position 270 by a polar        amino acid selected from the group consisting of WYSTCQNRKHD, in        particular DQ, notably D,    -   substitution of the tyrosine Y or leucine L in position 271 by a        non-bulky amino acid selected from the group consisting of        GPIVADCSTN or by a hydrophobic amino acid selected from the        group consisting of VIMFGAPWC, in particular VA,    -   substitution of the lysine K in position 272 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular MLA,    -   substitution of the proline P in position 273 by a non-bulky        amino acid selected from the group consisting of GLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular DEL, notably DL,    -   substitution of the lysine K in position 274 by a non-bulky        amino acid selected from the group consisting of GLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRHD, in particular R,    -   substitution of the proline P in position 277 by a bulky amino        acid selected from the group consisting of EHKRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAWYC, in particular KAV, notably K,    -   substitution of the arginine R or lysine K in position 278 by a        polar amino acid selected from the group consisting of        WYSTCQNHDE, in particular DNQ, notably DN,    -   substitution of the serine S in position 280 by a non-bulky        amino acid selected from the group consisting of GPLIVADCTN or        by a polar amino acid selected from the group consisting of        WYTCQNRKHDE, in particular GH,    -   substitution of the threonine T or alanine A or serine S in        position 282 by a non-bulky amino acid selected from the group        consisting of GPLIVDCN or by a hydrophobic amino acid selected        from the group consisting of VILMFGPWYC, in particular LV,        notably L,    -   and substitution of the isoleucine I in position 284 non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular VAL, notably V.

These 21 particular substitutions in position I168, D192, Y258, C260,T261, D263, G265, T/I266, A267, K/R268, P269, E270, Y/L271, K272, P273,K274, P277, R/K278, S280, T/A/S282 and 1284 belong to the second set ofsubstitutions called set 2.

These positions are mainly implicated in AHLs substrate accommodationwithin the active site of the enzyme. They were selected by analyzingthe evolutive history of this family of enzymes.

When set 2 is related to a sequence, it means that at least onesubstitution of said set occurs in said sequence.

The invention relates more particularly to the above-mentioned mutatedhyperthermophilic PTE having a lactonase activity, derived from ahyperthermophilic phosphotriesterase according to the present invention,wherein the amino acid in position 2 in SEQ ID NO: 1 is missing, andwherein the at least one mutation is selected from the group consistingof:

-   -   substitution of the proline P in position 68 by a non-bulky        amino acid selected from the group consisting of GLIVADCSTN, in        particular GAV,    -   substitution of the glycine G in position 226 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN, in        particular AVP,    -   and substitution of the tryptophan W in position 279 by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPYC or by a charged amino acid selected from the group        consisting of RKHDEC, in particular RKDI, notably R.

These 3 particular substitutions in position P68, G226 and W279 belongto the third set of substitutions called set 3.

These positions are highly suspected as being implicated in enzymaticactivities of the enzyme.

When set 3 is related to a sequence, it means that at least onesubstitution of said set occurs in said sequence.

The invention relates even more particularly to the above-mentionedmutated hyperthermophilic PTE having a lactonase activity, derived froma hyperthermophilic phosphotriesterase according to the presentinvention, wherein the amino acid in position 2 in SEQ ID NO: 1 ismissing, and wherein the at least one mutation selected from the groupconsisting of:

-   -   substitution of the glycine G in position 8 by a non-bulky amino        acid selected from the group consisting of PLIVADCSTN, in        particular STA, notably S,    -   substitution of the lysine K in position 9 by a charged amino        acid selected from the group consisting of RHDEC, in particular        EDR, notably E,    -   substitution of the valine V in position 28 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular GIFA, notably A,    -   substitution of the phenylalanine F in position 47 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN or by a polar amino acid selected from the group        consisting of WYSTCQNRKHDE, in particular LYW, notably L, or        substitution of the leucine L in position 47 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular YW,    -   substitution of the lysine K in position 55 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular IRL, notably I,    -   substitution of the threonine T in position 69 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSN, in        particular VAS, notably S,    -   substitution of the leucine L in position 73 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular CAMI, notably I,    -   substitution of the isoleucine I in position 77 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular PTV, notably T,    -   substitution of the valine V in position 84 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular AGI, notably A,    -   substitution of the tyrosine Y in position 98 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular FCLW, notably W,    -   substitution of the tyrosine Y in position 100 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular GEWF, notably F,    -   substitution of the isoleucine I in position 123 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular LAV, notably L,    -   substitution of the leucine L or serine S or asparagine N in        position 131 by a non-bulky amino acid selected from the group        consisting of GPIVADCT, in particular PT, notably P,    -   substitution of the aspartic acid D in position 142 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE, in        particular SET, notably T,    -   substitution of the lysine K in position 165 by a polar amino        acid selected from the group consisting of WYSTCNQRHDE, in        particular NQR, notably N, or substitution of the asparagine N        in position 165 by a polar amino acid selected from the group        consisting of WYSTCQRKHDE, in particular QR,    -   substitution of the glycine G in position 194 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCNQRKHDE, in particular ST, notably S,    -   substitution of the arginine R in position 224 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNKHDE, in particular CSTAH, notably AC,    -   substitution of the leucine L in position 227 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by apolar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular AIVH, notably V,    -   substitution of the leucine L in position 229 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VIMFGAPWYC, in particular IM, notably M,    -   substitution of the phenylalanine F in position 230 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN, in particular LTAS, notably S,    -   substitution of the leucine L in position 231 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN, in        particular PVA, notably P,    -   substitution of the cysteine C in position 259 by a hydrophobic        amino acid selected from the group consisting of VILMFGAPWY, in        particular YLIA, notably LA,    -   substitution of the isoleucine I in position 262 by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWYC, in particular FWC, notably F,    -   substitution of the tryptophane W in position 264 by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPYC or by a non-bulky amino acid selected from the group        consisting of GPLIVADCSTN, in particular ALMFCITV, notably F,        with the proviso that the tryptophane W in position 264 can not        be substituted by a phenylalanine F in SEQ ID NO: 3,    -   substitution of the leucine L in position 275 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular AVP, notably P,    -   substitution of the alanine A in position 276 by a hydrophobic        amino acid selected from the group consisting of VILMFGPWYC or        by a non-bulky amino acid selected from the group consisting of        GPLIVDCSTN, in particular NVMT, notably T,    -   substitution of the isoleucine I or methionine M in position 281        by a non-bulky amino acid selected from the group consisting of        GPLVADCSTN or by a hydrophobic amino acid selected from the        group consisting of VLFGAPWYC, in particular TYP, notably T,    -   substitution of the leucine L in position 283 by a bulky amino        acid selected from the group consisting of EKHRQYWFM, in        particular FMH, notably M,    -   and substitution of the asparagine N or serine S or threonine T        in position 298 by a polar amino acid selected from the group        consisting of WYCQRKHDE, notably Q, further comprises at least        one mutation selected from the group consisting of:    -   substitution of the isoleucine I in position 168 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VLMFGAPWYC, in particular VAL, notably V,    -   substitution of the aspartic acid D in position 192 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN or by a polar amino acid selected from the group        consisting of WYSTCQNRKHE, in particular ST, notably S,    -   substitution of the tyrosine Y position 258 by a non-bulky amino        acid selected from the group consisting of GPLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWC, in particular CSVW, notably C,    -   substitution of the cysteine C in position 260 a non-bulky amino        acid selected from the group consisting of GPLIVADSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWY, in particular SFWV, notably S,    -   substitution of the threonine T in position 261 a non-bulky        amino acid selected from the group consisting of GPLIVADCSN or        by a polar amino acid selected from the group consisting of        WYSCQNRKHE, in particular GH, notably G,    -   substitution of the aspartic acid D in position 263 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE or        by a non-bulky amino acid selected from the group consisting of        GPLIVACSTN, in particular SLH,    -   substitution of the glycine G in position 265 non-bulky amino        acid selected from the group consisting of PLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFAPWYC, in particular AVP,    -   substitution of the threonine T or isoleucine I in position 266        by a hydrophobic amino acid selected from the group consisting        of VLMFGAPWYC or by a non-bulky amino acid selected from the        group consisting of GPLVADCSN, in particular VWP, notably V,    -   substitution of the alanine A in position 267 by a polar amino        acid selected from the group consisting of WYSTCQNRKHDE, in        particular NQ, notably N,    -   substitution of the lysine K or arginine R in position 268 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN or by a hydrophobic amino acid selected from the        group consisting of VILMFGAPWYC, in particular IAP, notably IP,    -   substitution of the proline P in position 269 by a hydrophobic        amino acid selected from the group consisting of VILMFGACWY, in        particular MCL, notably M,    -   substitution of the glutamic acid E in position 270 by a polar        amino acid selected from the group consisting of WYSTCQNRKHD, in        particular DQ, notably D,    -   substitution of the tyrosine Y or leucine L in position 271 by a        non-bulky amino acid selected from the group consisting of        GPIVADCSTN or by a hydrophobic amino acid selected from the        group consisting of VIMFGAPWC, in particular VA,    -   substitution of the lysine K in position 272 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular MLA,    -   substitution of the proline P in position 273 by a non-bulky        amino acid selected from the group consisting of GLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular DEL, notably DL,    -   substitution of the lysine K in position 274 by a non-bulky        amino acid selected from the group consisting of GLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRHD, in particular R,    -   substitution of the proline P in position 277 by a bulky amino        acid selected from the group consisting of EHKRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAWYC, in particular KAV, notably K,    -   substitution of the arginine R or lysine K in position 278 by a        polar amino acid selected from the group consisting of        WYSTCQNHDE, in particular DNQ, notably DN,    -   substitution of the serine S in position 280 by a non-bulky        amino acid selected from the group consisting of GPLIVADCTN or        by a polar amino acid selected from the group consisting of        WYTCQNRKHDE, in particular GH,    -   substitution of the threonine T or alanine A or serine S in        position 282 by a non-bulky amino acid selected from the group        consisting of GPLIVDCN or by a hydrophobic amino acid selected        from the group consisting of VILMFGPWYC, in particular LV,        notably L,    -   and substitution of the isoleucine I in position 284 non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular VAL, notably V.

It means that at least one substitution among the 29 particularsubstitutions of set 1 in position G8, K9, V28, F/L47, K55, T69, L73,I77, V84, Y98, Y100, I123, L/S/N131, D142, N165, G194, R224, L227, L229,F230, L231, C259, 1262, W264, L275, A276, I/M281, L283 and N/S/T298 canbe associated with at least one substitution among the 21 particularsubstitutions of set 2 in position 1168, D192, Y258, C260, T261, D263,G265, T/I266, A267, K/R268, P269, E270, Y/L271, K272, P273, K274, P277,R/K278, S280, T/A/S282 and 1284.

The invention relates even more particularly to the above-mentionedmutated hyperthermophilic PTE having a lactonase activity, derived froma hyperthermophilic phosphotriesterase according to the presentinvention, wherein the amino acid in position 2 in SEQ ID NO: 1 ismissing, and wherein the at least one mutation selected from the groupconsisting of:

-   -   substitution of the glycine G in position 8 by a non-bulky amino        acid selected from the group consisting of PLIVADCSTN, in        particular STA, notably S,    -   substitution of the lysine K in position 9 by a charged amino        acid selected from the group consisting of RHDEC, in particular        EDR, notably E,    -   substitution of the valine V in position 28 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular GIFA, notably A,    -   substitution of the phenylalanine F in position 47 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN or by a polar amino acid selected from the group        consisting of WYSTCQNRKHDE, in particular LYW, notably L, or        substitution of the leucine L in position 47 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular YW,    -   substitution of the lysine K in position 55 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular IRL, notably I,    -   substitution of the threonine T in position 69 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSN, in        particular VAS, notably S,    -   substitution of the leucine L in position 73 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular CAMI, notably I,    -   substitution of the isoleucine I in position 77 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular PTV, notably T,    -   substitution of the valine V in position 84 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular AGI, notably A,    -   substitution of the tyrosine Y in position 98 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular FCLW, notably W,    -   substitution of the tyrosine Y in position 100 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular GEWF, notably F,    -   substitution of the isoleucine I in position 123 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular LAV, notably L,    -   substitution of the leucine L or serine S or asparagine N in        position 131 by a non-bulky amino acid selected from the group        consisting of GPIVADCT, in particular PT, notably P,    -   substitution of the aspartic acid D in position 142 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE, in        particular SET, notably T,    -   substitution of the lysine K in position 165 by a polar amino        acid selected from the group consisting of WYSTCNQRHDE, in        particular NQR, notably N, or substitution of the asparagine N        in position 165 by a polar amino acid selected from the group        consisting of WYSTCQRKHDE, in particular QR,    -   substitution of the glycine G in position 194 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCNQRKHDE, in particular ST, notably S,    -   substitution of the arginine R in position 224 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNKHDE, in particular CSTAH, notably AC,    -   substitution of the leucine L in position 227 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by apolar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular AIVH, notably V,    -   substitution of the leucine L in position 229 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VIMFGAPWYC, in particular IM, notably M,    -   substitution of the phenylalanine F in position 230 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN, in particular LTAS, notably S,    -   substitution of the leucine L in position 231 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN, in        particular PVA, notably P,    -   substitution of the cysteine C in position 259 by a hydrophobic        amino acid selected from the group consisting of VILMFGAPWY, in        particular YLIA, notably LA,    -   substitution of the isoleucine I in position 262 by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWYC, in particular FWC, notably F,    -   substitution of the tryptophane W in position 264 by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPYC or by a non-bulky amino acid selected from the group        consisting of GPLIVADCSTN, in particular ALMFCITV, notably F,        with the proviso that the tryptophane W in position 264 can not        be substituted by a phenylalanine F in SEQ ID NO: 3,    -   substitution of the leucine L in position 275 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular AVP, notably P,    -   substitution of the alanine A in position 276 by a hydrophobic        amino acid selected from the group consisting of VILMFGPWYC or        by a non-bulky amino acid selected from the group consisting of        GPLIVDCSTN, in particular NVMT, notably T,    -   substitution of the isoleucine I or methionine M in position 281        by a non-bulky amino acid selected from the group consisting of        GPLVADCSTN or by a hydrophobic amino acid selected from the        group consisting of VLFGAPWYC, in particular TYP, notably T,    -   substitution of the leucine L in position 283 by a bulky amino        acid selected from the group consisting of EKHRQYWFM, in        particular FMH, notably M,    -   and substitution of the asparagine N or serine S or threonine T        in position 298 by a polar amino acid selected from the group        consisting of WYCQRKHDE, notably Q, further comprises at least        one mutation selected from the group consisting of:    -   substitution of the proline P in position 68 by a non-bulky        amino acid selected from the group consisting of GLIVADCSTN, in        particular GAV,    -   substitution of the glycine G in position 226 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN, in        particular AVP,    -   and substitution of the tryptophan W in position 279 by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPYC or by a charged amino acid selected from the group        consisting of RKHDEC, in particular RKDI, notably R.

It means that at least one substitution among the 29 particularsubstitutions of set 1 in position G8, K9, V28, F/L47, K55, T69, L73,I77, V84, Y98, Y100, I123, L/S/N131, D142, N165, G194, R224, L227, L229,F230, L231, C259, I262, W264, L275, A276, I/M281, L283 and N/S/T298 canbe associated with at least one substitution among the 3 particularsubstitutions of set 3 in position P68, G226 and W279.

The invention relates even more particularly to the above-mentionedmutated hyperthermophilic PTE having a lactonase activity, derived froma hyperthermophilic phosphotriesterase according to the presentinvention, wherein the amino acid in position 2 in SEQ ID NO: 1 ismissing, and wherein the at least one mutation selected from the groupconsisting of:

-   -   substitution of the glycine G in position 8 by a non-bulky amino        acid selected from the group consisting of PLIVADCSTN, in        particular STA, notably S,    -   substitution of the lysine K in position 9 by a charged amino        acid selected from the group consisting of RHDEC, in particular        EDR, notably E,    -   substitution of the valine V in position 28 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular GIFA, notably A,    -   substitution of the phenylalanine F in position 47 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN or by a polar amino acid selected from the group        consisting of WYSTCQNRKHDE, in particular LYW, notably L, or        substitution of the leucine L in position 47 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular YW,    -   substitution of the lysine K in position 55 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular IRL, notably I,    -   substitution of the threonine T in position 69 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSN, in        particular VAS, notably S,    -   substitution of the leucine L in position 73 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular CAMI, notably I,    -   substitution of the isoleucine I in position 77 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular PTV, notably T,    -   substitution of the valine V in position 84 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular AGI, notably A,    -   substitution of the tyrosine Y in position 98 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular FCLW, notably W,    -   substitution of the tyrosine Y in position 100 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular GEWF, notably F,    -   substitution of the isoleucine I in position 123 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular LAV, notably L,    -   substitution of the leucine L or serine S or asparagine N in        position 131 by a non-bulky amino acid selected from the group        consisting of GPIVADCT, in particular PT, notably P,    -   substitution of the aspartic acid D in position 142 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE, in        particular SET, notably T,    -   substitution of the lysine K in position 165 by a polar amino        acid selected from the group consisting of WYSTCNQRHDE, in        particular NQR, notably N, or substitution of the asparagine N        in position 165 by a polar amino acid selected from the group        consisting of WYSTCQRKHDE, in particular QR,    -   substitution of the glycine G in position 194 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCNQRKHDE, in particular ST, notably S,    -   substitution of the arginine R in position 224 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNKHDE, in particular CSTAH, notably AC,    -   substitution of the leucine L in position 227 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by apolar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular AIVH, notably V,    -   substitution of the leucine L in position 229 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VIMFGAPWYC, in particular IM, notably M,    -   substitution of the phenylalanine F in position 230 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN, in particular LTAS, notably S,    -   substitution of the leucine L in position 231 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN, in        particular PVA, notably P,    -   substitution of the cysteine C in position 259 by a hydrophobic        amino acid selected from the group consisting of VILMFGAPWY, in        particular YLIA, notably LA,    -   substitution of the isoleucine I in position 262 by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWYC, in particular FWC, notably F,    -   substitution of the tryptophane W in position 264 by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPYC or by a non-bulky amino acid selected from the group        consisting of GPLIVADCSTN, in particular ALMFCITV, notably F,        with the proviso that the tryptophane W in position 264 can not        be substituted by a phenylalanine F in SEQ ID NO: 3,    -   substitution of the leucine L in position 275 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular AVP, notably P,    -   substitution of the alanine A in position 276 by a hydrophobic        amino acid selected from the group consisting of VILMFGPWYC or        by a non-bulky amino acid selected from the group consisting of        GPLIVDCSTN, in particular NVMT, notably T,    -   substitution of the isoleucine I or methionine M in position 281        by a non-bulky amino acid selected from the group consisting of        GPLVADCSTN or by a hydrophobic amino acid selected from the        group consisting of VLFGAPWYC, in particular TYP, notably T,    -   substitution of the leucine L in position 283 by a bulky amino        acid selected from the group consisting of EKHRQYWFM, in        particular FMH, notably M,    -   and substitution of the asparagine N or serine S or threonine T        in position 298 by a polar amino acid selected from the group        consisting of WYCQRKHDE, notably Q, further comprises at least        one mutation selected from the group consisting of:    -   substitution of the isoleucine I in position 168 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VLMFGAPWYC, in particular VAL, notably V,    -   substitution of the aspartic acid D in position 192 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN or by a polar amino acid selected from the group        consisting of WYSTCQNRKHE, in particular ST, notably S,    -   substitution of the tyrosine Y position 258 by a non-bulky amino        acid selected from the group consisting of GPLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWC, in particular CSVW, notably C,    -   substitution of the cysteine C in position 260 a non-bulky amino        acid selected from the group consisting of GPLIVADSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWY, in particular SFWV, notably S,    -   substitution of the threonine T in position 261 a non-bulky        amino acid selected from the group consisting of GPLIVADCSN or        by a polar amino acid selected from the group consisting of        WYSCQNRKHE, in particular GH, notably G,    -   substitution of the aspartic acid D in position 263 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE or        by a non-bulky amino acid selected from the group consisting of        GPLIVACSTN, in particular SLH,    -   substitution of the glycine G in position 265 non-bulky amino        acid selected from the group consisting of PLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFAPWYC, in particular AVP,    -   substitution of the threonine T or isoleucine I in position 266        by a hydrophobic amino acid selected from the group consisting        of VLMFGAPWYC or by a non-bulky amino acid selected from the        group consisting of GPLVADCSN, in particular VWP, notably V,    -   substitution of the alanine A in position 267 by a polar amino        acid selected from the group consisting of WYSTCQNRKHDE, in        particular NQ, notably N,    -   substitution of the lysine K or arginine R in position 268 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN or by a hydrophobic amino acid selected from the        group consisting of VILMFGAPWYC, in particular IAP, notably IP,    -   substitution of the proline P in position 269 by a hydrophobic        amino acid selected from the group consisting of VILMFGACWY, in        particular MCL, notably M,    -   substitution of the glutamic acid E in position 270 by a polar        amino acid selected from the group consisting of WYSTCQNRKHD, in        particular DQ, notably D,    -   substitution of the tyrosine Y or leucine L in position 271 by a        non-bulky amino acid selected from the group consisting of        GPIVADCSTN or by a hydrophobic amino acid selected from the        group consisting of VIMFGAPWC, in particular VA,    -   substitution of the lysine K in position 272 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular MLA,    -   substitution of the proline P in position 273 by a non-bulky        amino acid selected from the group consisting of GLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular DEL, notably DL,    -   substitution of the lysine K in position 274 by a non-bulky        amino acid selected from the group consisting of GLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRHD, in particular R,    -   substitution of the proline P in position 277 by a bulky amino        acid selected from the group consisting of EHKRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAWYC, in particular KAV, notably K,    -   substitution of the arginine R or lysine K in position 278 by a        polar amino acid selected from the group consisting of        WYSTCQNHDE, in particular DNQ, notably DN,    -   substitution of the serine S in position 280 by a non-bulky        amino acid selected from the group consisting of GPLIVADCTN or        by a polar amino acid selected from the group consisting of        WYTCQNRKHDE, in particular GH,    -   substitution of the threonine T or alanine A or serine S in        position 282 by a non-bulky amino acid selected from the group        consisting of GPLIVDCN or by a hydrophobic amino acid selected        from the group consisting of VILMFGPWYC, in particular LV,        notably L,    -   and substitution of the isoleucine I in position 284 non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular VAL, notably V, further comprises at least one        mutation selected from the group consisting of:    -   substitution of the proline P in position 68 by a non-bulky        amino acid selected from the group consisting of GLIVADCSTN, in        particular GAV,    -   substitution of the glycine G in position 226 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN, in        particular AVP,    -   and substitution of the tryptophan W in position 279 by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPYC or by a charged amino acid selected from the group        consisting of RKHDEC, in particular RKDI, notably R.

It means that at the at least one substitution among the 29 particularsubstitutions of set 1 in position G8, K9, V28, F/L47, K55, T69, L73,I77, V84, Y98, Y100, I123, L/S/N131, D142, N165, G194, R224, L227, L229,F230, L231, C259, 1262, W264, L275, A276, I/M281, L283 and N/S/T298 canbe associated with at least one substitution among the 21 particularsubstitutions of set 2 in position I168, D192, Y258, C260, T261, D263,G265, T/I266, A267, K/R268, P269, E270, Y/L271, K272, P273, K274, P277,R/K278, S280, T/A/S282 and 1284 and with at least one substitution amongthe 3 particular substitutions of set 3 in position P68, G226 and W279.

The invention relates even more particularly to the above-mentionedmutated hyperthermophilic PTE having a lactonase activity, derived froma hyperthermophilic phosphotriesterase according to the presentinvention, wherein the amino acid in position 2 in SEQ ID NO: 1 ismissing, and wherein the at least one mutation selected from the groupconsisting of:

-   -   substitution of the isoleucine I in position 168 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VLMFGAPWYC, in particular VAL, notably V,    -   substitution of the aspartic acid D in position 192 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN or by a polar amino acid selected from the group        consisting of WYSTCQNRKHE, in particular ST, notably S,    -   substitution of the tyrosine Y position 258 by a non-bulky amino        acid selected from the group consisting of GPLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWC, in particular CSVW, notably C,    -   substitution of the cysteine C in position 260 a non-bulky amino        acid selected from the group consisting of GPLIVADSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWY, in particular SFWV, notably S,    -   substitution of the threonine T in position 261 a non-bulky        amino acid selected from the group consisting of GPLIVADCSN or        by a polar amino acid selected from the group consisting of        WYSCQNRKHE, in particular GH, notably G,    -   substitution of the aspartic acid D in position 263 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE or        by a non-bulky amino acid selected from the group consisting of        GPLIVACSTN, in particular SLH,    -   substitution of the glycine G in position 265 non-bulky amino        acid selected from the group consisting of PLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFAPWYC, in particular AVP,    -   substitution of the threonine T or isoleucine I in position 266        by a hydrophobic amino acid selected from the group consisting        of VLMFGAPWYC or by a non-bulky amino acid selected from the        group consisting of GPLVADCSN, in particular VWP, notably V,    -   substitution of the alanine A in position 267 by a polar amino        acid selected from the group consisting of WYSTCQNRKHDE, in        particular NQ, notably N,    -   substitution of the lysine K or arginine R in position 268 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN or by a hydrophobic amino acid selected from the        group consisting of VILMFGAPWYC, in particular IAP, notably IP,    -   substitution of the proline P in position 269 by a hydrophobic        amino acid selected from the group consisting of VILMFGACWY, in        particular MCL, notably M,    -   substitution of the glutamic acid E in position 270 by a polar        amino acid selected from the group consisting of WYSTCQNRKHD, in        particular DQ, notably D,    -   substitution of the tyrosine Y or leucine L in position 271 by a        non-bulky amino acid selected from the group consisting of        GPIVADCSTN or by a hydrophobic amino acid selected from the        group consisting of VIMFGAPWC, in particular VA,    -   substitution of the lysine K in position 272 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular MLA,    -   substitution of the proline P in position 273 by a non-bulky        amino acid selected from the group consisting of GLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular DEL, notably DL,    -   substitution of the lysine K in position 274 by a non-bulky        amino acid selected from the group consisting of GLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRHD, in particular R,    -   substitution of the proline P in position 277 by a bulky amino        acid selected from the group consisting of EHKRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAWYC, in particular KAV, notably K,    -   substitution of the arginine R or lysine K in position 278 by a        polar amino acid selected from the group consisting of        WYSTCQNHDE, in particular DNQ, notably DN,    -   substitution of the serine S in position 280 by a non-bulky        amino acid selected from the group consisting of GPLIVADCTN or        by a polar amino acid selected from the group consisting of        WYTCQNRKHDE, in particular GH,    -   substitution of the threonine T or alanine A or serine S in        position 282 by a non-bulky amino acid selected from the group        consisting of GPLIVDCN or by a hydrophobic amino acid selected        from the group consisting of VILMFGPWYC, in particular LV,        notably L,    -   and substitution of the isoleucine I in position 284 non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular VAL, notably V, further comprises at least one        mutation selected from the group consisting of:    -   substitution of the proline P in position 68 by a non-bulky        amino acid selected from the group consisting of GLIVADCSTN, in        particular GAV,    -   substitution of the glycine G in position 226 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN, in        particular AVP,    -   and substitution of the tryptophan W in position 279 by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPYC or by a charged amino acid selected from the group        consisting of RKHDEC, in particular RKDI, notably R.

It means that at the at least one substitution among the 21 particularsubstitutions of set 2 in position I168, D192, Y258, C260, T261, D263,G265, T/I266, A267, K/R268, P269, E270, Y/L271, K272, P273, K274, P277,R/K278, S280, T/A/S282 and 1284 can be associated with at least onesubstitution among the 3 particular substitutions of set 3 in positionP68, G226 and W279.

The invention relates more particularly to the above-mentioned mutatedhyperthermophilic PTEs having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus solfataricus corresponding to thesequence SEQ ID NO: 3, said mutated PTEs comprising the at least onemutation selected from the group consisting of: substitutions of thevaline V in position 27, the proline P in position 67, the threonine Tin position 68, the leucine L in position 72, the tyrosine Y in position97, the tyrosine Y in position 99, the aspartic acid D in position 141,the arginine R in position 223, the glycine G in position 225, theleucine L in position 226, the phenylalanine F in position 229, thecysteine C in position 258, the tryptophane W in position 263 and thetryptophan W in position 278, of SEQ ID NO: 3 by any other natural ornon-natural amino acid.

In an even more specific embodiment, the above-mentioned mutatedhyperthermophilic PTEs having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus solfataricus corresponding to thesequence SEQ ID NO: 3, comprise only one mutation, said mutation beingselected from the group consisting of: substitutions of the valine V inposition 27, the proline P in position 67, the threonine T in position68, the leucine L in position 72, the aspartic acid D in position 141,the glycine G in position 225, the leucine L in position 226, thephenylalanine F in position 229, the tryptophane W in position 263 andthe tryptophan W in position 278, of SEQ ID NO: 3 by any other naturalor non-natural amino acid.

In a more specific embodiment, the above-mentioned mutatedhyperthermophilic PTEs having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus solfataricus corresponding to thesequence SEQ ID NO: 3, comprise the at least one mutation selected fromthe group consisting of: substitutions of the glycine G in position 7,the lysine K in position 8, the phenylalanine F in position 46, thelysine K in position 54, the isoleucine I in position 76, the valine Vin position 83, the isoleucine I in position 122, the leucine L inposition 130, the lysine K in position 164, the isoleucine I in position167, the aspartic acid D in position 191, the glycine G in position 193,the leucine L in position 228, the leucine L in position 230, thetyrosine Y position 257, the cysteine C in position 259, the threonine Tin position 260, the isoleucine I in position 261, the aspartic acid Din position 262, the glycine G in position 264, the threonine T inposition 265, the alanine A in position 266, the lysine K in position267, the proline P in position 268, the glutamic acid E in position 269,the tyrosine Y in position 270, the lysine K in position 271, theproline P in position 272, the lysine K in position 273, the leucine Lin position 274, the alanine A in position 275, the proline P inposition 276, the arginine R in position 277, the serine S in position279, the isoleucine I in position 280, the threonine T in position 281,the leucine L in position 282, the isoleucine I in position 283 and theasparagine N in position 297, of SEQ ID NO: 3 by any other natural ornon-natural amino acid.

In an even more specific embodiment, the above-mentioned mutatedhyperthermophilic PTEs having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus solfataricus corresponding to thesequence SEQ ID NO: 3, comprise only one mutation, said mutation beingselected from the group consisting of: substitutions of the glycine G inposition 7, the lysine K in position 8, the phenylalanine F in position46, the lysine K in position 54, the isoleucine I in position 76, thevaline V in position 83, the isoleucine I in position 122, the leucine Lin position 130, the lysine K in position 164, the isoleucine I inposition 167, the aspartic acid D in position 191, the glycine G inposition 193, the leucine L in position 228, the leucine L in position230, the tyrosine Y position 257, the cysteine C in position 259, thethreonine T in position 260, the isoleucine I in position 261, theaspartic acid D in position 262, the glycine G in position 264, thethreonine T in position 265, the alanine A in position 266, the lysine Kin position 267, the proline P in position 268, the glutamic acid E inposition 269, the tyrosine Y in position 270, the lysine K in position271, the proline P in position 272, the lysine K in position 273, theleucine L in position 274, the alanine A in position 275, the proline Pin position 276, the arginine R in position 277, the serine S inposition 279, the isoleucine I in position 280, the threonine T inposition 281, the leucine L in position 282, the isoleucine I inposition 283 and the asparagine N in position 297, of SEQ ID NO: 3 byany other natural or non-natural amino acid.

The invention relates more particularly to the above-mentioned mutatedhyperthermophilic PTE having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus solfataricus corresponding to thesequence SEQ ID NO: 3, and wherein the at least one mutation is selectedfrom the group consisting of:

-   -   substitution of the glycine G in position 7 by a non-bulky amino        acid selected from the group consisting of PLIVADCSTN, in        particular STA, notably S,    -   substitution of the lysine K in position 8 by a charged amino        acid selected from the group consisting of RHDEC, in particular        EDR, notably E,    -   substitution of the valine V in position 27 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular GIFA, notably A,    -   substitution of the phenylalanine F in position 46 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN or by a polar amino acid selected from the group        consisting of WYSTCQNRKHDE, in particular LYW, notably L,    -   substitution of the lysine K in position 54 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular IRL, notably I,    -   substitution of the threonine T in position 68 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSN, in        particular VAS, notably S,    -   substitution of the leucine L in position 72 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular CAMI, notably I,    -   substitution of the isoleucine I in position 76 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular PTV, notably T,    -   substitution of the valine V in position 83 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular AGI, notably A,    -   substitution of the tyrosine Y in position 97 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular FCLW, notably W,    -   substitution of the tyrosine Y in position 99 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular GEWF, notably F,    -   substitution of the isoleucine I in position 122 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular LAV, notably L,    -   substitution of the leucine L in position 130 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN, in        particular PST, notably P,    -   substitution of the aspartic acid D in position 141 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE, in        particular SET, notably T,    -   substitution of the lysine K in position 164 by a polar amino        acid selected from the group consisting of WYSTCQNRHDE, in        particular NQR, notably N,    -   substitution of the glycine G in position 193 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCNQRKHDE, in particular ST, notably S,    -   substitution of the arginine R in position 223 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNKHDE, in particular CSTAH, notably AC,    -   substitution of the leucine L in position 226 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by apolar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular AIVH, notably V,    -   substitution of the leucine L in position 228 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VIMFGAPWYC, in particular IM, notably M,    -   substitution of the phenylalanine F in position 229 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN, in particular LTAS, notably S,    -   substitution of the leucine L in position 230 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN, in        particular PVA, notably P,    -   substitution of the cysteine C in position 258 by a hydrophobic        amino acid selected from the group consisting of VILMFGAPWY, in        particular YLIA, notably LA,    -   substitution of the isoleucine I in position 261 by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWYC, in particular FWC, notably F,    -   substitution of the tryptophane W in position 263 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMGAPYC, in particular ALMCITV,    -   substitution of the leucine L in position 274 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular AVP, notably P,    -   substitution of the alanine A in position 275 by a hydrophobic        amino acid selected from the group consisting of VILMFGPWYC or        by a non-bulky amino acid selected from the group consisting of        GPLIVDCSTN, in particular NVMT, notably T,    -   substitution of the isoleucine I in position 280 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VLMFGAPWYC, in particular TMYP, notably T,    -   substitution of the leucine L in position 282 by a bulky amino        acid selected from the group consisting of EKHRQYWFM, in        particular FMH, notably M,    -   and substitution of the asparagine N in position 297 by a polar        amino acid selected from the group consisting of WYSTCQRKHDE, in        particular QST, notably S.

These 29 particular substitutions in position G7, K8, V27, F46, K54,T68, L72, I76, V83, Y97, Y99, I122, L130, D141, N164, G193, R223, L226,L228, F229, L230, C258, I261, W263, L274, A275, I280, L282 and N297belong to the four set of substitutions called set 4.

The invention relates more particularly to the above-mentioned mutatedhyperthermophilic PTE having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus solfataricus corresponding to thesequence SEQ ID NO: 3, and wherein the at least one mutation is selectedfrom the group consisting of:

-   -   substitution of the isoleucine I in position 167 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VLMFGAPWYC, in particular VAL, notably V,    -   substitution of the aspartic acid D in position 191 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN or by a polar amino acid selected from the group        consisting of WYSTCQNRKHE, in particular ST, notably S,    -   substitution of the tyrosine Y position 257 by a non-bulky amino        acid selected from the group consisting of GPLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWC, in particular CSVW, notably C,    -   substitution of the cysteine C in position 259 a non-bulky amino        acid selected from the group consisting of GPLIVADSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWY, in particular SFWV, notably S,    -   substitution of the threonine T in position 260 a non-bulky        amino acid selected from the group consisting of GPLIVADCSN or        by a polar amino acid selected from the group consisting of        WYSCQNRKHE, in particular GH, notably G,    -   substitution of the aspartic acid D in position 262 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE or        by a non-bulky amino acid selected from the group consisting of        GPLIVADCSTN, in particular SLH,    -   substitution of the glycine G in position 264 non-bulky amino        acid selected from the group consisting of PLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFAPWYC, in particular AVP,    -   substitution of the threonine T in position 265 by a hydrophobic        amino acid selected from the group consisting of VILMFGAPWYC or        by a non-bulky amino acid selected from the group consisting of        GPLIVADCSN, in particular VWP, notably V,    -   substitution of the alanine A in position 266 by a polar amino        acid selected from the group consisting of WYSTCQNRKHDE, in        particular NQ, notably N,    -   substitution of the lysine K in position 267 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWYC, in particular IAP, notably IP,    -   substitution of the proline P in position 268 by a hydrophobic        amino acid selected from the group consisting of VILMFGAWYC, in        particular MCL, notably M,    -   substitution of the glutamic acid E in position 269 by a polar        amino acid selected from the group consisting of WYSTCQNRKHD, in        particular DQ, notably D,    -   substitution of the tyrosine Y in position 270 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWC, in particular VAL,    -   substitution of the lysine K in position 271 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular MLA,    -   substitution of the proline P in position 272 by a non-bulky        amino acid selected from the group consisting of GLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular DEL, notably DL,    -   substitution of the lysine K in position 273 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRHD, in particular RP,    -   substitution of the proline P in position 276 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAWYC, in particular KAV, notably K,    -   substitution of the arginine R in position 277 by a polar amino        acid selected from the group consisting of WYSTCQNKHDE, in        particular DNQ, notably DN,    -   substitution of the serine S in position 279 by a non-bulky        amino acid selected from the group consisting of GPLIVADCTN or        by a polar amino acid selected from the group consisting of        WYTCQNRKHDE, in particular GH,    -   substitution of the threonine T in position 281 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWYC, in particular ALV, notably AL,    -   and substitution of the isoleucine I in position 283 non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular VAL, notably V.

These 21 particular substitutions in position I167, D191, Y257, C259,T260, D262, G264, T265, A266, K267, P268, E269, Y270, K271, P272, K273,P276, R277, S279, T281 and 1283 belong to the fifth set of substitutionscalled set 5.

The invention relates more particularly to the above-mentioned mutatedhyperthermophilic PTE having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus solfataricus corresponding to thesequence SEQ ID NO: 3, and wherein the at least one mutation is selectedfrom the group consisting of:

-   -   substitution of the proline P in position 67 by a non-bulky        amino acid selected from the group consisting of GLIVADCSTN, in        particular GAV,    -   substitution of the glycine G in position 225 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN, in        particular AVP,    -   and substitution of the tryptophan W in position 278 by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPYC or by a charged amino acid selected from the group        consisting of RKHDEC, in particular RKDI, notably R.

These 3 particular substitutions in position P67, G225 and W278 belongto the sixth set of substitutions called set 6.

The invention also relates to the above-mentioned mutatedhyperthermophilic PTE having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus solfataricus corresponding to thesequence SEQ ID NO: 3, wherein the at least one mutation selected fromthe group consisting of:

-   -   substitution of the glycine G in position 7 by a non-bulky amino        acid selected from the group consisting of PLIVADCSTN, in        particular STA, notably S,    -   substitution of the lysine K in position 8 by a charged amino        acid selected from the group consisting of RHDEC, in particular        EDR, notably E,    -   substitution of the valine V in position 27 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular GIFA, notably A,    -   substitution of the phenylalanine F in position 46 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN or by a polar amino acid selected from the group        consisting of WYSTCQNRKHDE, in particular LYW, notably L,    -   substitution of the lysine K in position 54 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular IRL, notably I,    -   substitution of the threonine T in position 68 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSN, in        particular VAS, notably S,    -   substitution of the leucine L in position 72 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular CAMI, notably I,    -   substitution of the isoleucine I in position 76 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular PTV, notably T,    -   substitution of the valine V in position 83 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular AGI, notably A,    -   substitution of the tyrosine Y in position 97 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular FCLW, notably W,    -   substitution of the tyrosine Y in position 99 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular GEWF, notably F,    -   substitution of the isoleucine I in position 122 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular LAV, notably L,    -   substitution of the leucine L in position 130 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN, in        particular PST, notably P,    -   substitution of the aspartic acid D in position 141 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE, in        particular SET, notably T,    -   substitution of the lysine K in position 164 by a polar amino        acid selected from the group consisting of WYSTCQNRHDE, in        particular NQR, notably N,    -   substitution of the glycine G in position 193 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCNQRKHDE, in particular ST, notably S,    -   substitution of the arginine R in position 223 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNKHDE, in particular CSTAH, notably AC,    -   substitution of the leucine L in position 226 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by apolar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular AIVH, notably V,    -   substitution of the leucine L in position 228 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VIMFGAPWYC, in particular IM, notably M,    -   substitution of the phenylalanine F in position 229 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN, in particular LTAS, notably S,    -   substitution of the leucine L in position 230 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN, in        particular PVA, notably P,    -   substitution of the cysteine C in position 258 by a hydrophobic        amino acid selected from the group consisting of VILMFGAPWY, in        particular YLIA, notably LA,    -   substitution of the isoleucine I in position 261 by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWYC, in particular FWC, notably F,    -   substitution of the tryptophane W in position 263 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMGAPYC, in particular ALMCITV,    -   substitution of the leucine L in position 274 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular AVP, notably P,    -   substitution of the alanine A in position 275 by a hydrophobic        amino acid selected from the group consisting of VILMFGPWYC or        by a non-bulky amino acid selected from the group consisting of        GPLIVDCSTN, in particular NVMT, notably T,    -   substitution of the isoleucine I in position 280 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VLMFGAPWYC, in particular TMYP, notably T,    -   substitution of the leucine L in position 282 by a bulky amino        acid selected from the group consisting of EKHRQYWFM, in        particular FMH, notably M,    -   and substitution of the asparagine N in position 297 by a polar        amino acid selected from the group consisting of WYSTCQRKHDE, in        particular QST, notably S, further comprises at least one        mutation selected from the group consisting of:    -   substitution of the isoleucine I in position 167 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VLMFGAPWYC, in particular VAL, notably V,    -   substitution of the aspartic acid D in position 191 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN or by a polar amino acid selected from the group        consisting of WYSTCQNRKHE, in particular ST, notably S,    -   substitution of the tyrosine Y position 257 by a non-bulky amino        acid selected from the group consisting of GPLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWC, in particular CSVW, notably C,    -   substitution of the cysteine C in position 259 a non-bulky amino        acid selected from the group consisting of GPLIVADSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWY, in particular SFWV, notably S,    -   substitution of the threonine T in position 260 a non-bulky        amino acid selected from the group consisting of GPLIVADCSN or        by a polar amino acid selected from the group consisting of        WYSCQNRKHE, in particular GH, notably G,    -   substitution of the aspartic acid D in position 262 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE or        by a non-bulky amino acid selected from the group consisting of        GPLIVADCSTN, in particular SLH,    -   substitution of the glycine G in position 264 non-bulky amino        acid selected from the group consisting of PLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFAPWYC, in particular AVP,    -   substitution of the threonine T in position 265 by a hydrophobic        amino acid selected from the group consisting of VILMFGAPWYC or        by a non-bulky amino acid selected from the group consisting of        GPLIVADCSN, in particular VWP, notably V,    -   substitution of the alanine A in position 266 by a polar amino        acid selected from the group consisting of WYSTCQNRKHDE, in        particular NQ, notably N,    -   substitution of the lysine K in position 267 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWYC, in particular IAP, notably IP,    -   substitution of the proline P in position 268 by a hydrophobic        amino acid selected from the group consisting of VILMFGAWYC, in        particular MCL, notably M,    -   substitution of the glutamic acid E in position 269 by a polar        amino acid selected from the group consisting of WYSTCQNRKHD, in        particular DQ, notably D,    -   substitution of the tyrosine Y in position 270 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWC, in particular VAL,    -   substitution of the lysine K in position 271 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular MLA,    -   substitution of the proline P in position 272 by a non-bulky        amino acid selected from the group consisting of GLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular DEL, notably DL,    -   substitution of the lysine K in position 273 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRHD, in particular RP,    -   substitution of the proline P in position 276 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAWYC, in particular KAV, notably K,    -   substitution of the arginine R in position 277 by a polar amino        acid selected from the group consisting of WYSTCQNKHDE, in        particular DNQ, notably DN,    -   substitution of the serine S in position 279 by a non-bulky        amino acid selected from the group consisting of GPLIVADCTN or        by a polar amino acid selected from the group consisting of        WYTCQNRKHDE, in particular GH,    -   substitution of the threonine T in position 281 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWYC, in particular ALV, notably AL,    -   and substitution of the isoleucine I in position 283 non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular VAL, notably V.

It means that at least one substitution among the 29 particularsubstitutions of set 4 in position G7, K8, V27, F46, K54, T68, L72, I76,V83, Y97, Y99, I122, L130, D141, N164, G193, R223, L226, L228, F229,L230, C258, I261, W263, L274, A275, I280, L282 and N297 can beassociated with at least one substitution among the 21 particularsubstitutions of set 5 in position I167, D191, Y257, C259, T260, D262,G264, T265, A266, K267, P268, E269, Y270, K271, P272, K273, P276, R277,S279, T281.

The invention also relates to the above-mentioned mutatedhyperthermophilic PTE having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus solfataricus corresponding to thesequence SEQ ID NO: 3, wherein the at least one mutation selected fromthe group consisting of:

-   -   substitution of the glycine G in position 7 by a non-bulky amino        acid selected from the group consisting of PLIVADCSTN, in        particular STA, notably S,    -   substitution of the lysine K in position 8 by a charged amino        acid selected from the group consisting of RHDEC, in particular        EDR, notably E,    -   substitution of the valine V in position 27 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular GIFA, notably A,    -   substitution of the phenylalanine F in position 46 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN or by a polar amino acid selected from the group        consisting of WYSTCQNRKHDE, in particular LYW, notably L,    -   substitution of the lysine K in position 54 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular IRL, notably I,    -   substitution of the threonine T in position 68 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSN, in        particular VAS, notably S,    -   substitution of the leucine L in position 72 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular CAMI, notably I,    -   substitution of the isoleucine I in position 76 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular PTV, notably T,    -   substitution of the valine V in position 83 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular AGI, notably A,    -   substitution of the tyrosine Y in position 97 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular FCLW, notably W,    -   substitution of the tyrosine Y in position 99 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular GEWF, notably F,    -   substitution of the isoleucine I in position 122 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular LAV, notably L,    -   substitution of the leucine L in position 130 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN, in        particular PST, notably P,    -   substitution of the aspartic acid D in position 141 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE, in        particular SET, notably T,    -   substitution of the lysine K in position 164 by a polar amino        acid selected from the group consisting of WYSTCQNRHDE, in        particular NQR, notably N,    -   substitution of the glycine G in position 193 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCNQRKHDE, in particular ST, notably S,    -   substitution of the arginine R in position 223 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNKHDE, in particular CSTAH, notably AC,    -   substitution of the leucine L in position 226 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by apolar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular AIVH, notably V,    -   substitution of the leucine L in position 228 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VIMFGAPWYC, in particular IM, notably M,    -   substitution of the phenylalanine F in position 229 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN, in particular LTAS, notably S,    -   substitution of the leucine L in position 230 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN, in        particular PVA, notably P,    -   substitution of the cysteine C in position 258 by a hydrophobic        amino acid selected from the group consisting of VILMFGAPWY, in        particular YLIA, notably LA,    -   substitution of the isoleucine I in position 261 by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWYC, in particular FWC, notably F,    -   substitution of the tryptophane W in position 263 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMGAPYC, in particular ALMCITV,    -   substitution of the leucine L in position 274 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular AVP, notably P,    -   substitution of the alanine A in position 275 by a hydrophobic        amino acid selected from the group consisting of VILMFGPWYC or        by a non-bulky amino acid selected from the group consisting of        GPLIVDCSTN, in particular NVMT, notably T,    -   substitution of the isoleucine I in position 280 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VLMFGAPWYC, in particular TMYP, notably T,    -   substitution of the leucine L in position 282 by a bulky amino        acid selected from the group consisting of EKHRQYWFM, in        particular FMH, notably M,    -   and substitution of the asparagine N in position 297 by a polar        amino acid selected from the group consisting of WYSTCQRKHDE, in        particular QST, notably S, further comprises at least one        mutation selected from the group consisting of:    -   substitution of the proline P in position 67 by a non-bulky        amino acid selected from the group consisting of GLIVADCSTN, in        particular GAV,    -   substitution of the glycine G in position 225 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN, in        particular AVP,    -   and substitution of the tryptophan W in position 278 by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPYC or by a charged amino acid selected from the group        consisting of RKHDEC, in particular RKDI, notably R.

It means that at least one substitution among the 29 particularsubstitutions of set 4 in position G7, K8, V27, F46, K54, T68, L72, 176,V83, Y97, Y99, I122, L130, D141, N164, G193, R223, L226, L228, F229,L230, C258, 1261, W263, L274, A275, I280, L282 and N297 can beassociated with at least one substitution among the 3 particularsubstitutions of set 6 in position P67, G225 and W278.

The invention also relates to the above-mentioned mutatedhyperthermophilic PTE having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus solfataricus corresponding to thesequence SEQ ID NO: 3, wherein the at least one mutation selected fromthe group consisting of:

-   -   substitution of the glycine G in position 7 by a non-bulky amino        acid selected from the group consisting of PLIVADCSTN, in        particular STA, notably S,    -   substitution of the lysine K in position 8 by a charged amino        acid selected from the group consisting of RHDEC, in particular        EDR, notably E,    -   substitution of the valine V in position 27 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular GIFA, notably A,    -   substitution of the phenylalanine F in position 46 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN or by a polar amino acid selected from the group        consisting of WYSTCQNRKHDE, in particular LYW, notably L,    -   substitution of the lysine K in position 54 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular IRL, notably I,    -   substitution of the threonine T in position 68 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSN, in        particular VAS, notably S,    -   substitution of the leucine L in position 72 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular CAMI, notably I,    -   substitution of the isoleucine I in position 76 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular PTV, notably T,    -   substitution of the valine V in position 83 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular AGI, notably A,    -   substitution of the tyrosine Y in position 97 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular FCLW, notably W,    -   substitution of the tyrosine Y in position 99 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular GEWF, notably F,    -   substitution of the isoleucine I in position 122 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular LAV, notably L,    -   substitution of the leucine L in position 130 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN, in        particular PST, notably P,    -   substitution of the aspartic acid D in position 141 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE, in        particular SET, notably T,    -   substitution of the lysine K in position 164 by a polar amino        acid selected from the group consisting of WYSTCQNRHDE, in        particular NQR, notably N,    -   substitution of the glycine G in position 193 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCNQRKHDE, in particular ST, notably S,    -   substitution of the arginine R in position 223 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNKHDE, in particular CSTAH, notably AC,    -   substitution of the leucine L in position 226 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by apolar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular AIVH, notably V,    -   substitution of the leucine L in position 228 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VIMFGAPWYC, in particular IM, notably M,    -   substitution of the phenylalanine F in position 229 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN, in particular LTAS, notably S,    -   substitution of the leucine L in position 230 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN, in        particular PVA, notably P,    -   substitution of the cysteine C in position 258 by a hydrophobic        amino acid selected from the group consisting of VILMFGAPWY, in        particular YLIA, notably LA,    -   substitution of the isoleucine I in position 261 by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWYC, in particular FWC, notably F,    -   substitution of the tryptophane W in position 263 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMGAPYC, in particular ALMCITV,    -   substitution of the leucine L in position 274 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular AVP, notably P,    -   substitution of the alanine A in position 275 by a hydrophobic        amino acid selected from the group consisting of VILMFGPWYC or        by a non-bulky amino acid selected from the group consisting of        GPLIVDCSTN, in particular NVMT, notably T,    -   substitution of the isoleucine I in position 280 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VLMFGAPWYC, in particular TMYP, notably T,    -   substitution of the leucine L in position 282 by a bulky amino        acid selected from the group consisting of EKHRQYWFM, in        particular FMH, notably M,    -   and substitution of the asparagine N in position 297 by a polar        amino acid selected from the group consisting of WYSTCQRKHDE, in        particular QST, notably S, further comprises at least one        mutation selected from the group consisting of:    -   substitution of the isoleucine I in position 167 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VLMFGAPWYC, in particular VAL, notably V,    -   substitution of the aspartic acid D in position 191 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN or by a polar amino acid selected from the group        consisting of WYSTCQNRKHE, in particular ST, notably S,    -   substitution of the tyrosine Y position 257 by a non-bulky amino        acid selected from the group consisting of GPLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWC, in particular CSVW, notably C,    -   substitution of the cysteine C in position 259 a non-bulky amino        acid selected from the group consisting of GPLIVADSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWY, in particular SFWV, notably S,    -   substitution of the threonine T in position 260 a non-bulky        amino acid selected from the group consisting of GPLIVADCSN or        by a polar amino acid selected from the group consisting of        WYSCQNRKHE, in particular GH, notably G,    -   substitution of the aspartic acid D in position 262 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE or        by a non-bulky amino acid selected from the group consisting of        GPLIVADCSTN, in particular SLH,    -   substitution of the glycine G in position 264 non-bulky amino        acid selected from the group consisting of PLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFAPWYC, in particular AVP,    -   substitution of the threonine T in position 265 by a hydrophobic        amino acid selected from the group consisting of VILMFGAPWYC or        by a non-bulky amino acid selected from the group consisting of        GPLIVADCSN, in particular VWP, notably V,    -   substitution of the alanine A in position 266 by a polar amino        acid selected from the group consisting of WYSTCQNRKHDE, in        particular NQ, notably N,    -   substitution of the lysine K in position 267 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWYC, in particular IAP, notably IP,    -   substitution of the proline P in position 268 by a hydrophobic        amino acid selected from the group consisting of VILMFGAWYC, in        particular MCL, notably M,    -   substitution of the glutamic acid E in position 269 by a polar        amino acid selected from the group consisting of WYSTCQNRKHD, in        particular DQ, notably D,    -   substitution of the tyrosine Y in position 270 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWC, in particular VAL,    -   substitution of the lysine K in position 271 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular MLA,    -   substitution of the proline P in position 272 by a non-bulky        amino acid selected from the group consisting of GLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular DEL, notably DL,    -   substitution of the lysine K in position 273 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRHD, in particular RP,    -   substitution of the proline P in position 276 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAWYC, in particular KAV, notably K,    -   substitution of the arginine R in position 277 by a polar amino        acid selected from the group consisting of WYSTCQNKHDE, in        particular DNQ, notably DN,    -   substitution of the serine S in position 279 by a non-bulky        amino acid selected from the group consisting of GPLIVADCTN or        by a polar amino acid selected from the group consisting of        WYTCQNRKHDE, in particular GH,    -   substitution of the threonine T in position 281 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWYC, in particular ALV, notably AL,    -   and substitution of the isoleucine I in position 283 non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular VAL, notably V, further comprises at least one        mutation selected from the group consisting of:    -   substitution of the proline P in position 67 by a non-bulky        amino acid selected from the group consisting of GLIVADCSTN, in        particular GAV,    -   substitution of the glycine G in position 225 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN, in        particular AVP,    -   and substitution of the tryptophan W in position 278 by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPYC or by a charged amino acid selected from the group        consisting of RKHDEC, in particular RKDI, notably R.

It means that at least one substitution among the 29 particularsubstitutions of set 4 in position G7, K8, V27, F46, K54, T68, L72, 176,V83, Y97, Y99, I122, L130, D141, N164, G193, R223, L226, L228, F229,L230, C258, 1261, W263, L274, A275, I280, L282 and N297 can beassociated with at least one substitution among the 21 particularsubstitutions of set 5 in position I167, D191, Y257, C259, T260, D262,G264, T265, A266, K267, P268, E269, Y270, K271, P272, K273, P276, R277,S279, T281 and with at least one substitution among the 3 particularsubstitutions of set 6 in position P67, G225 and W278.

The invention also relates to the above-mentioned mutatedhyperthermophilic PTE having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus solfataricus corresponding to thesequence SEQ ID NO: 3, wherein the at least one mutation selected fromthe group consisting of:

-   -   substitution of the isoleucine I in position 167 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VLMFGAPWYC, in particular VAL, notably V,    -   substitution of the aspartic acid D in position 191 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN or by a polar amino acid selected from the group        consisting of WYSTCQNRKHE, in particular ST, notably S,    -   substitution of the tyrosine Y position 257 by a non-bulky amino        acid selected from the group consisting of GPLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWC, in particular CSVW, notably C,    -   substitution of the cysteine C in position 259 a non-bulky amino        acid selected from the group consisting of GPLIVADSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWY, in particular SFWV, notably S,    -   substitution of the threonine T in position 260 a non-bulky        amino acid selected from the group consisting of GPLIVADCSN or        by a polar amino acid selected from the group consisting of        WYSCQNRKHE, in particular GH, notably G,    -   substitution of the aspartic acid D in position 262 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE or        by a non-bulky amino acid selected from the group consisting of        GPLIVADCSTN, in particular SLH,    -   substitution of the glycine G in position 264 non-bulky amino        acid selected from the group consisting of PLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFAPWYC, in particular AVP,    -   substitution of the threonine T in position 265 by a hydrophobic        amino acid selected from the group consisting of VILMFGAPWYC or        by a non-bulky amino acid selected from the group consisting of        GPLIVADCSN, in particular VWP, notably V,    -   substitution of the alanine A in position 266 by a polar amino        acid selected from the group consisting of WYSTCQNRKHDE, in        particular NQ, notably N,    -   substitution of the lysine K in position 267 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWYC, in particular IAP, notably IP,    -   substitution of the proline P in position 268 by a hydrophobic        amino acid selected from the group consisting of VILMFGAWYC, in        particular MCL, notably M,    -   substitution of the glutamic acid E in position 269 by a polar        amino acid selected from the group consisting of WYSTCQNRKHD, in        particular DQ, notably D,    -   substitution of the tyrosine Y in position 270 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWC, in particular VAL,    -   substitution of the lysine K in position 271 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular MLA,    -   substitution of the proline P in position 272 by a non-bulky        amino acid selected from the group consisting of GLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular DEL, notably DL,    -   substitution of the lysine K in position 273 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRHD, in particular RP,    -   substitution of the proline P in position 276 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAWYC, in particular KAV, notably K,    -   substitution of the arginine R in position 277 by a polar amino        acid selected from the group consisting of WYSTCQNKHDE, in        particular DNQ, notably DN,    -   substitution of the serine S in position 279 by a non-bulky        amino acid selected from the group consisting of GPLIVADCTN or        by a polar amino acid selected from the group consisting of        WYTCQNRKHDE, in particular GH,    -   substitution of the threonine T in position 281 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWYC, in particular ALV, notably AL,    -   and substitution of the isoleucine I in position 283 non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular VAL, notably V, further comprises at least one        mutation selected from the group consisting of:    -   substitution of the proline P in position 67 by a non-bulky        amino acid selected from the group consisting of GLIVADCSTN, in        particular GAV,    -   substitution of the glycine G in position 225 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN, in        particular AVP,    -   and substitution of the tryptophan W in position 278 by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPYC or by a charged amino acid selected from the group        consisting of RKHDEC, in particular RKDI, notably R.

It means that the at least one substitution among the 21 particularsubstitutions of set 5 in position I167, D191, Y257, C259, T260, D262,G264, T265, A266, K267, P268, E269, Y270, K271, P272, K273, P276, R277,S279, T281 can be associated with at least one substitution among the 3particular substitutions of set 6 in position P67, G225 and W278.

A more particular subject of the invention is mutated hyperthermophilicPTE having a lactonase activity according to the present invention,derived from the hyperthermophilic PTE of Sulfolobus solfataricuscorresponding to the sequence SEQ ID NO: 3, said mutatedhyperthermophilic PTE correspond to the following sequences:

-   -   SEQ ID NO: 9 corresponding to the SEQ ID NO: 3 comprising the        following one mutation: substitution of the tryptophan W in        position 263 by a methionine M,    -   SEQ ID NO: 11 corresponding to the SEQ ID NO: 3 comprising the        following one mutation: substitution of the tryptophan W in        position 263 by a leucine L,    -   SEQ ID NO: 13 corresponding to the SEQ ID NO: 3 comprising the        following one mutation: substitution of the tryptophan W in        position 263 by an alanine A,    -   SEQ ID NO: 15 corresponding to the SEQ ID NO: 3 comprising the        following one mutation: substitution of the tryptophan W in        position 263 by an isoleucine I,    -   SEQ ID NO: 17 corresponding to the SEQ ID NO: 3 comprising the        following one mutation: substitution of the tryptophan W in        position 263 by a valine V,    -   SEQ ID NO: 19 corresponding to the SEQ ID NO: 3 comprising the        following one mutation: substitution of the tryptophan W in        position 263 by a threonine T,    -   SEQ ID NO: 21 corresponding to the SEQ ID NO: 3 comprising the        following three mutations: substitution of the cysteine C in        position 258 by a leucine L, substitution of the isoleucine I in        position 261 by a phenylalanine F, substitution of the        tryptophan W in position 263 by an alanine A,    -   SEQ ID NO: 23 corresponding to the SEQ ID NO: 3 comprising the        following four mutations: substitution of the valine V in        position 27 by an alanine A, substitution of the tyrosine Y in        position 97 by a tryptophan W, substitution of the leucine L in        position 228 by a methionine M, substitution of the tryptophan W        in position 263 by a methionine M,    -   SEQ ID NO: 25 corresponding to the SEQ ID NO: 3 comprising the        following four mutations: substitution of the valine V in        position 27 by an alanine A, substitution of the tyrosine Y in        position 97 by a tryptophan W, substitution of the tryptophan W        in position 263 by a leucine L, substitution of the isoleucine I        in position 280 by a threonine T,    -   SEQ ID NO: 27 corresponding to the SEQ ID NO: 3 comprising the        following four mutations: substitution of the phenylalanine F in        position 46 by a leucine L, substitution of the cytosine C in        position 258 by an alanine A, substitution of the tryptophan W        in position 263 by a methionine M, substitution of the        isoleucine I in position 280 by a threonine T,    -   SEQ ID NO: 29 corresponding to the SEQ ID NO: 3 comprising the        following six mutations: substitution of the valine V in        position 27 by an alanine A, substitution of the isoleucine I in        position 76 by a threonine T, substitution of the tyrosine Y in        position 97 by a tryptophan W, substitution of the tyrosine Y in        position 99 by a phenylalanine F, substitution of the leucine L        in position 130 by a proline P, substitution of the leucine L in        position 226 by a valine V,    -   SEQ ID NO: 31 corresponding to the SEQ ID NO: 3 comprising the        following six mutations: substitution of the leucine L in        position 72 by an isoleucine I, substitution of the tyrosine Y        in position 99 by a phenylalanine F, substitution of the        isoleucine I in position 122 by a leucine L, substitution of the        leucine L in position 228 by a methionine M, substitution of the        phenylalanine F in position 229 by a serine S, substitution of        the tryptophan W in position 263 by a leucine L,    -   SEQ ID NO: 33 corresponding to the SEQ ID NO: 3 comprising the        following seven mutations: substitution of the threonine T in        position 68 by a serine S, substitution of the leucine L in        position 72 by an isoleucine I, substitution of the leucine L in        position 130 by a proline P, substitution of the leucine L in        position 228 by a methionine M, substitution of the        phenylalanine F in position 229 by a serine S, substitution of        the tryptophan W in position 263 by a methionine M, substitution        of the leucine L in position 274 by a proline P,    -   SEQ ID NO: 35 corresponding to the SEQ ID NO: 3 comprising the        following six mutations: substitution of the threonine T in        position 68 by a serine S, substitution of the isoleucine I in        position 76 by a threonine T, substitution of the tyrosine Y in        position 97 by a tryptophan W, substitution of the tyrosine Y in        position 99 by a phenylalanine F, substitution of the leucine L        in position 228 by a methionine M, substitution of the        tryptophan W in position 263 by a leucine L,    -   SEQ ID NO: 37 corresponding to the SEQ ID NO: 3 comprising the        following five mutations: substitution of the lysine K in        position 8 by an glutamic acid E, substitution of the        phenylalanine F in position 46 by a leucine L, substitution of        the tyrosine Y in position 97 by a tryptophan W, substitution of        the tyrosine Y in position 99 by a phenylalanine F, substitution        of the leucine L in position 228 by a methionine M,    -   SEQ ID NO: 39 corresponding to the SEQ ID NO: 3 comprising the        following two mutations: substitution of the leucine L in        position 72 by an isoleucine I, substitution of the tryptophan W        in position 263 by a phenylalanine F,    -   SEQ ID NO: 41 corresponding to the SEQ ID NO: 3 comprising the        following five mutations: substitution of the threonine T in        position 68 by a serine S, substitution of the tyrosine Y in        position 97 by a tryptophan W, substitution of the tyrosine Y in        position 99 by a phenylalanine F, substitution of the leucine L        in position 130 by a proline P, substitution of the leucine L in        position 228 by a methionine M,    -   SEQ ID NO: 43 corresponding to the SEQ ID NO: 3 comprising the        following four mutations: substitution of the valine V in        position 27 by an alanine A, substitution of the phenylalanine F        in position 46 by a leucine L, substitution of the leucine L in        position 226 by a valine V, substitution the tryptophan W in        position 263 by a leucine L,    -   SEQ ID NO: 45 corresponding to the SEQ ID NO: 3 comprising the        following eight mutations: substitution of the proline P in        position 67 by a valine V, substitution of the threonine T in        position 68 by a serine S, substitution of the tyrosine Y in        position 97 by a tryptophan W, substitution of the tyrosine Y in        position 99 by a phenylalanine F, substitution of the leucine L        in position 228 by a methionine M, substitution of the cysteine        C in position 258 by an alanine A, substitution the tryptophan W        in position 263 by a leucine L, substitution of the isoleucine I        in position 280 by a threonine T,    -   SEQ ID NO: 47 corresponding to the SEQ ID NO: 3 comprising the        following eight mutations: substitution of the phenylalanine F        in position 46 by a leucine L, substitution of the threonine T        in position 68 by a serine S, substitution of the tyrosine Y in        position 97 by a tryptophan W, substitution of the tyrosine Y in        position 99 by a phenylalanine F, substitution of the leucine L        in position 130 by a proline P, substitution of the lysine K in        position 164 by an asparagine N, substitution of the leucine L        in position 226 by a valine V, substitution the tryptophan W in        position 263 by a methionine M,    -   SEQ ID NO: 49 corresponding to the SEQ ID NO: 3 comprising the        following five mutations: substitution of the threonine T in        position 68 by a serine S, substitution of the leucine L in        position 72 by an isoleucine I, substitution of the tyrosine Y        in position 97 by a tryptophan W, substitution of the tyrosine Y        in position 99 by a phenylalanine F, substitution of the leucine        L in position 130 by a proline P.

The coding sequence of the above-mentioned mutated hyperthermophilic PTEhaving a lactonase activity according to the present invention, derivedfrom the hyperthermophilic PTE of Sulfolobus solfataricus correspondingto the sequence SEQ ID NO: 3 and corresponding to the followingsequences SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, and 48 are also part of the invention.

The invention also related to mutated hyperthermophilic PTE having alactonase activity according to the present invention, derived from thehyperthermophilic PTE of Sulfolobus solfataricus corresponding to thesequence SEQ ID NO: 3, said mutated hyperthermophilic PTE correspond tothe following sequences SEQ ID NO: 139, 141, 143, 145, 147, 149, 151,153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177 and 179for the proteins and to their respective coding sequences SEQ ID NO:138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,166, 168, 170, 172, 174, 176 and 178.

A more particular subject of the invention is the above-mentionedmutated hyperthermophilic PTE having a lactonase activity, in which atleast one of the amino acids involved in the salt bridges is modified bysubstitution, or deletion, such that the activation temperature of saidmutated hyperthermophilic PTE having a lactonase activity is reducedcompared with the activation temperature of the mutatedhyperthermophilic PTE having a lactonase activity in which the aminoacids involved in the salt bridges is unmodified.

By “susbstitution” is meant the replacement of an amino acid by another.By “deletion” is meant the removal of an amino acid, such that theprotein sequence which has bveen subjected to said deletion is shorterthan the sequence wich has not been subjected to said deletion.

In a preferred embodiment, the amino acids involved in the salt bridgesmentioned previously can be replaced by a sequence of at least two aminoacids. This is then an “addition” and the the protein sequence which hasbeen subjected to said addition is longer than the sequence wich has notbeen subjected to said addition.

The substitutions defined according to the invention relate equally tonatural or non-natural (artificial) amino acids. Thus, the amino acidsinvolved in salt bridges can be replaced by a natural or an artificialamino acid.

The invention relates more particularly to the above-mentioned mutatedhyperthermophilic PTE having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus solfataricus corresponding to thesequence SEQ ID NO: 3, further comprising at least one mutationcorresponding to a substitution of at least one of the amino acids ofthe following amino acid pairs, the positions of which in SEQ ID NO: 3are indicated hereafter, by another natural or non-natural amino acid:2R/314S, 14K/12E, 26R/75D, 26R/42E, 33R/42E, 33R/45E, 55R/52E, 55R/285E,74R/121D, 81K/42E, 81K/43D, 84K/80E, 109R/113E, 123K/162E, 147K/148D,151K/148D, 154R/150E, 154R/187E, 154R/188E, 161K/188E, 183R/150E,183R/187E, 183R/180E, 210K/245D, 215K/214D, 223R/256D, 223R/202D,234K/204D, 235R/202D, 241R/245D, 245D/244K, 250K/249D, 277R/286D,292K/298E, 310K/307E.

The invention relates also to the above-mentioned mutatedhyperthermophilic PTEs having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus acidocaldarius corresponding to thesequence SEQ ID NO: 5, said mutated PTEs comprising the at least onemutation selected from the group consisting of: substitutions of theglycine G in position 8, the lysine K in position 9, the valine V inposition 28, the leucine L in position 47, the lysine K in position 55,the proline P in position 68, the threonine T in position 69, theleucine L in position 73, the isoleucine I in position 77, the valine Vin position 84, the tyrosine Y in position 98, the tyrosine Y inposition 100, the isoleucine I in position 123, the asparagine N inposition 131, the aspartic acid D in position 142, the asparagine N inposition 165, the isoleucine I in position 168, the aspartic acid D inposition 192, the glycine G in position 194, the arginine R in position224, the glycine G in position 226, the leucine L in position 227, theleucine L in position 229, the phenylalanine F in position 230, theleucine L in position 231, the tyrosine Y position 258, the cysteine Cin position 259, the cysteine C in position 260, the threonine T inposition 261, the isoleucine I in position 262, the aspartic acid D inposition 263, the tryptophane W in position 264, the glycine G inposition 265, the isoleucine I in position 266, the alanine A inposition 267, the lysine K in position 268, the proline P in position269, the glutamic acid E in position 270, the tyrosine Y in position271, the lysine K in position 272, the proline P in position 273, thelysine K in position 274, the leucine L in position 275, the alanine Ain position 276, the proline P in position 277, the lysine K in position278, the tryptophan W in position 279, the serine S in position 280, themethionine M in position 281, the serine S in position 282, the leucineL in position 283, the isoleucine I in position 284 and the threonine Tin position 298, of SEQ ID NO: 5 by any other natural or non-naturalamino acid.

In a more specific embodiment, the above-mentioned mutatedhyperthermophilic PTEs having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus acidocaldarius corresponding to thesequence SEQ ID NO: 5, comprise the at least one mutation selected fromthe group consisting of: substitutions of the valine V in position 28,the proline P in position 68, the threonine T in position 69, theleucine L in position 73, the tyrosine Y in position 98, the tyrosine Yin position 100, the aspartic acid D in position 142, the arginine R inposition 224, the glycine G in position 226, the leucine L in position227, the phenylalanine F in position 230, the cysteine C in position259, the tryptophane W in position 264 and the tryptophan W in position279, of SEQ ID NO: 5 by any other natural or non-natural amino acid.

In an even more specific embodiment, the above-mentioned mutatedhyperthermophilic PTEs having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus acidocaldarius corresponding to thesequence SEQ ID NO: 5, comprise only one mutation, said mutation beingselected from the group consisting of: substitutions of the valine V inposition 28, the proline P in position 68, the threonine T in position69, the leucine L in position 73, the aspartic acid D in position 142,the glycine G in position 226, the leucine L in position 227, thephenylalanine F in position 230, the tryptophane W in position 264, thetryptophan W in position 279, of SEQ ID NO: 5 by any other natural ornon-natural amino acid.

In a more specific embodiment, the above-mentioned mutatedhyperthermophilic PTEs having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus acidocaldarius corresponding to thesequence SEQ ID NO: 5, comprise the at least one mutation selected fromthe group consisting of: substitutions of the glycine G in position 8,the lysine K in position 9, the leucine L in position 47, the lysine Kin position 55, the isoleucine I in position 77, the valine V inposition 84, the isoleucine I in position 123, the asparagine N inposition 131, the asparagine N in position 165, the isoleucine I inposition 168, the aspartic acid D in position 192, the glycine G inposition 194, the leucine L in position 229, the leucine L in position231, the tyrosine Y position 258, the cysteine C in position 260, thethreonine T in position 261, the isoleucine I in position 262, theaspartic acid D in position 263, the glycine G in position 265, theisoleucine I in position 266, the alanine A in position 267, the lysineK in position 268, the proline P in position 269, the glutamic acid E inposition 270, the tyrosine Y in position 271, the lysine K in position272, the proline P in position 273, the lysine K in position 274, theleucine L in position 275, the alanine A in position 276, the proline Pin position 277, the lysine K in position 278, the serine S in position280, the methionine M in position 281, the serine S in position 282, theleucine L in position 283, the isoleucine I in position 284 and thethreonine T in position 298, of SEQ ID NO: 5 by any other natural ornon-natural amino acid.

In an even more specific embodiment, the above-mentioned mutatedhyperthermophilic PTEs having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus acidocaldarius corresponding to thesequence SEQ ID NO: 5, comprise only one mutation, said mutation beingselected from the group consisting of: substitutions of the glycine G inposition 8, the lysine K in position 9, the leucine L in position 47,the lysine K in position 55, the isoleucine I in position 77, the valineV in position 84, the isoleucine I in position 123, the asparagine N inposition 131, the asparagine N in position 165, the isoleucine I inposition 168, the aspartic acid D in position 192, the glycine G inposition 194, the leucine L in position 229, the leucine L in position231, the tyrosine Y position 258, the cysteine C in position 260, thethreonine T in position 261, the isoleucine I in position 262, theaspartic acid D in position 263, the glycine G in position 265, theisoleucine I in position 266, the alanine A in position 267, the lysineK in position 268, the proline P in position 269, the glutamic acid E inposition 270, the tyrosine Y in position 271, the lysine K in position272, the proline P in position 273, the lysine K in position 274, theleucine L in position 275, the alanine A in position 276, the proline Pin position 277, the lysine K in position 278, the serine S in position280, the methionine M in position 281, the serine S in position 282, theleucine L in position 283, the isoleucine I in position 284, thethreonine T in position 298, of SEQ ID NO: 5 by any other natural ornon-natural amino acid.

The invention relates more particularly to the above-mentioned mutatedhyperthermophilic PTE having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus acidocaldarius corresponding to thesequence SEQ ID NO: 5, and wherein the at least one mutation is selectedfrom the group consisting of:

-   -   substitution of the glycine G in position 8 by a non-bulky amino        acid selected from the group consisting of PLIVADCSTN, in        particular STA, notably S,    -   substitution of the lysine K in position 9 by a charged amino        acid selected from the group consisting of RHDEC, in particular        EDR, notably E,    -   substitution of the valine V in position 28 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular GIFA, notably A,    -   substitution of the leucine L in position 47 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular YW,    -   substitution of the lysine K in position 55 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular IRL, notably I,    -   substitution of the threonine T in position 69 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSN, in        particular VAS, notably S,    -   substitution of the leucine L in position 73 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular CAMI, notably I,    -   substitution of the isoleucine I in position 77 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular PTV, notably T,    -   substitution of the valine V in position 84 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular AGI, notably A,    -   substitution of the tyrosine Y in position 98 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular FCLW, notably W,    -   substitution of the tyrosine Y in position 100 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular GEWF, notably F,    -   substitution of the isoleucine I in position 123 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular LAV, notably L,    -   substitution of the asparagine N in position 131 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN, in        particular PST, notably P,    -   substitution of the aspartic acid D in position 142 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE, in        particular SET, notably T,    -   substitution of the asparagine N in position 165 by a polar        amino acid selected from the group consisting of WYSTCQRKHDE, in        particular QR,    -   substitution of the glycine G in position 194 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCNQRKHDE, in particular ST, notably S,    -   substitution of the arginine R in position 224 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNKHDE, in particular CSTAH, notably AC,    -   substitution of the leucine L in position 227 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by apolar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular AIVH, notably V,    -   substitution of the leucine L in position 229 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VIMFGAPWYC, in particular IM, notably M,    -   substitution of the phenylalanine F in position 230 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN, in particular LTAS, notably S,    -   substitution of the leucine L in position 231 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN, in        particular PVA, notably P,    -   substitution of the cysteine C in position 259 by a hydrophobic        amino acid selected from the group consisting of VILMFGAPWY, in        particular YLIA, notably LA,    -   substitution of the isoleucine I in position 262 by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWYC, in particular FWC, notably F,    -   substitution of the tryptophane W in position 264 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPYC, in particular ALMFCITV, notably F,    -   substitution of the leucine L in position 275 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular AVP, notably P,    -   substitution of the alanine A in position 276 by a hydrophobic        amino acid selected from the group consisting of VILMFGPWYC or        by a non-bulky amino acid selected from the group consisting of        GPLIVDCSTN, in particular NVMT, notably T,    -   substitution of the methionine M in position 281 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILFGAPWYC, in particular TYP, notably T,    -   substitution of the leucine L in position 283 by a bulky amino        acid selected from the group consisting of EKHRQYWFM, in        particular FMH, notably M,    -   and substitution of the threonine T in position 298 by a polar        amino acid selected from the group consisting of WYSCQNRKHDE, in        particular QS, notably S.

These 29 particular substitutions in position G8, K9, V28, L47, K55,T69, L73, I77, V84, Y98, Y100, I123, N131, D142, N165, G194, R224, L227,L229, F230, L231, C259, 1262, W264, L275, A276, M281, L283 and T298belong to the seventh set of substitutions called set 7.

The invention relates more particularly to the above-mentioned mutatedhyperthermophilic PTE having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus acidocaldarius corresponding to thesequence SEQ ID NO: 5, and wherein the at least one mutation is selectedfrom the group consisting of:

-   -   substitution of the isoleucine I in position 168 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VLMFGAPWYC, in particular VAL, notably V,    -   substitution of the aspartic acid D in position 192 by a        non-bulky amino acid selected from the group consisting of        GPLVADCSTN or by a polar amino acid selected from the group        consisting of WYSTCQNRKHE, in particular ST, notably S,    -   substitution of the tyrosine Y position 258 by a non-bulky amino        acid selected from the group consisting of GPLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWC, in particular CSVW, notably C,    -   substitution of the cysteine C in position 260 a non-bulky amino        acid selected from the group consisting of GPLIVADSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWY, in particular SFWV, notably S,    -   substitution of the threonine T in position 261 a non-bulky        amino acid selected from the group consisting of GPLIVADCSN or        by a polar amino acid selected from the group consisting of        WYSCQNRKHE, in particular GH, notably G,    -   substitution of the aspartic acid D in position 263 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE or        by a non-bulky amino acid selected from the group consisting of        GPLIVACSTN, in particular SLH,    -   substitution of the glycine G in position 265 non-bulky amino        acid selected from the group consisting of PLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFAPWYC, in particular AVP,    -   substitution of the isoleucine I in position 266 by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWYC or by a non-bulky amino acid selected from the group        consisting of GPLVADCSTN, in particular VWP, notably V,    -   substitution of the alanine A in position 267 by a polar amino        acid selected from the group consisting of WYSTCQNRKHDE, in        particular NQ, notably N,    -   substitution of the lysine K in position 268 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWYC, in particular IAP, notably IP,    -   substitution of the proline P in position 269 by a hydrophobic        amino acid selected from the group consisting of VILMFGAWYC, in        particular MCL, notably M,    -   substitution of the glutamic acid E in position 270 by a polar        amino acid selected from the group consisting of WYSTCQNRKHD, in        particular DQ, notably D,    -   substitution of the tyrosine Y in position 271 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWC, in particular VAL,    -   substitution of the lysine K in position 272 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular MLA,    -   substitution of the proline P in position 273 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular DEL, notably DL,    -   substitution of the lysine K in position 274 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRHD, in particular RP,    -   substitution of the proline P in position 277 by a bulky amino        acid selected from the group consisting of EHKRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAWYC, in particular KAV, notably K,    -   substitution of the lysine K in position 278 by a polar amino        acid selected from the group consisting of WYSTCQNRHDE, in        particular DNQ, notably DN,    -   substitution of the serine S in position 280 by a non-bulky        amino acid selected from the group consisting of GPLIVADCTN or        by a polar amino acid selected from the group consisting of        WYTCQNRKHDE, in particular GH,    -   substitution of the serine S in position 282 by a non-bulky        amino acid selected from the group consisting of GPLIVADCTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWYC, in particular ALV, notably AL,    -   and substitution of the isoleucine I in position 284 non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular VAL, notably V.

These 21 particular substitutions in position 1168, D192, Y258, C260,T261, D263, G265, I266, A267, K268, P269, E270, Y/L271, K272, P273,K274, P277, K278, S280, S282 and 1284 belong to the eighth set ofsubstitutions called set 8.

The invention relates more particularly to the above-mentioned mutatedhyperthermophilic PTE having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus acidocaldarius corresponding to thesequence SEQ ID NO: 5, and wherein the at least one mutation is selectedfrom the group consisting of:

-   -   substitution of the proline P in position 68 by a non-bulky        amino acid selected from the group consisting of GLIVADCSTN, in        particular GAV,    -   substitution of the glycine G in position 226 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN, in        particular AVP,    -   and substitution of the tryptophan W in position 279 by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPYC or by a charged amino acid selected from the group        consisting of RKHDEC, in particular RKDI, notably R. These 3        particular substitutions in position P68, G226 and W279 belong        to the ninth set of substitutions called set 9.

The invention also relates to the above-mentioned mutatedhyperthermophilic PTE having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus acidocaldarius corresponding to thesequence SEQ ID NO: 5, and wherein the at least one mutation selectedfrom the group consisting of:

-   -   substitution of the glycine G in position 8 by a non-bulky amino        acid selected from the group consisting of PLIVADCSTN, in        particular STA, notably S,    -   substitution of the lysine K in position 9 by a charged amino        acid selected from the group consisting of RHDEC, in particular        EDR, notably E,    -   substitution of the valine V in position 28 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular GIFA, notably A,    -   substitution of the leucine L in position 47 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular YW,    -   substitution of the lysine K in position 55 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular IRL, notably I,    -   substitution of the threonine T in position 69 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSN, in        particular VAS, notably S,    -   substitution of the leucine L in position 73 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular CAMI, notably I,    -   substitution of the isoleucine I in position 77 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular PTV, notably T,    -   substitution of the valine V in position 84 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular AGI, notably A,    -   substitution of the tyrosine Y in position 98 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular FCLW, notably W,    -   substitution of the tyrosine Y in position 100 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular GEWF, notably F,    -   substitution of the isoleucine I in position 123 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular LAV, notably L,    -   substitution of the asparagine N in position 131 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN, in        particular PST, notably P,    -   substitution of the aspartic acid D in position 142 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE, in        particular SET, notably T,    -   substitution of the asparagine N in position 165 by a polar        amino acid selected from the group consisting of WYSTCQRKHDE, in        particular QR,    -   substitution of the glycine G in position 194 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCNQRKHDE, in particular ST, notably S,    -   substitution of the arginine R in position 224 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNKHDE, in particular CSTAH, notably AC,    -   substitution of the leucine L in position 227 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by apolar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular AIVH, notably V,    -   substitution of the leucine L in position 229 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VIMFGAPWYC, in particular IM, notably M,    -   substitution of the phenylalanine F in position 230 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN, in particular LTAS, notably S,    -   substitution of the leucine L in position 231 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN, in        particular PVA, notably P,    -   substitution of the cysteine C in position 259 by a hydrophobic        amino acid selected from the group consisting of VILMFGAPWY, in        particular YLIA, notably LA,    -   substitution of the isoleucine I in position 262 by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWYC, in particular FWC, notably F,    -   substitution of the tryptophane W in position 264 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPYC, in particular ALMFCITV, notably F,    -   substitution of the leucine L in position 275 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular AVP, notably P,    -   substitution of the alanine A in position 276 by a hydrophobic        amino acid selected from the group consisting of VILMFGPWYC or        by a non-bulky amino acid selected from the group consisting of        GPLIVDCSTN, in particular NVMT, notably T,    -   substitution of the methionine M in position 281 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILFGAPWYC, in particular TYP, notably T,    -   substitution of the leucine L in position 283 by a bulky amino        acid selected from the group consisting of EKHRQYWFM, in        particular FMH, notably M,    -   and substitution of the threonine T in position 298 by a polar        amino acid selected from the group consisting of WYSCQNRKHDE, in        particular QS, notably S, further comprises at least one        mutation selected from the group consisting of:    -   substitution of the isoleucine I in position 168 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VLMFGAPWYC, in particular VAL, notably V,    -   substitution of the aspartic acid D in position 192 by a        non-bulky amino acid selected from the group consisting of        GPLVADCSTN or by a polar amino acid selected from the group        consisting of WYSTCQNRKHE, in particular ST, notably S,    -   substitution of the tyrosine Y position 258 by a non-bulky amino        acid selected from the group consisting of GPLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWC, in particular CSVW, notably C,    -   substitution of the cysteine C in position 260 a non-bulky amino        acid selected from the group consisting of GPLIVADSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWY, in particular SFWV, notably S,    -   substitution of the threonine T in position 261 a non-bulky        amino acid selected from the group consisting of GPLIVADCSN or        by a polar amino acid selected from the group consisting of        WYSCQNRKHE, in particular GH, notably G,    -   substitution of the aspartic acid D in position 263 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE or        by a non-bulky amino acid selected from the group consisting of        GPLIVACSTN, in particular SLH,    -   substitution of the glycine G in position 265 non-bulky amino        acid selected from the group consisting of PLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFAPWYC, in particular AVP,    -   substitution of the isoleucine I in position 266 by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWYC or by a non-bulky amino acid selected from the group        consisting of GPLVADCSTN, in particular VWP, notably V,    -   substitution of the alanine A in position 267 by a polar amino        acid selected from the group consisting of WYSTCQNRKHDE, in        particular NQ, notably N,    -   substitution of the lysine K in position 268 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWYC, in particular IAP, notably IP,    -   substitution of the proline P in position 269 by a hydrophobic        amino acid selected from the group consisting of VILMFGAWYC, in        particular MCL, notably M,    -   substitution of the glutamic acid E in position 270 by a polar        amino acid selected from the group consisting of WYSTCQNRKHD, in        particular DQ, notably D,    -   substitution of the tyrosine Y in position 271 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWC, in particular VAL,    -   substitution of the lysine K in position 272 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular MLA,    -   substitution of the proline P in position 273 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular DEL, notably DL,    -   substitution of the lysine K in position 274 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRHD, in particular RP,    -   substitution of the proline P in position 277 by a bulky amino        acid selected from the group consisting of EHKRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAWYC, in particular KAV, notably K,    -   substitution of the lysine K in position 278 by a polar amino        acid selected from the group consisting of WYSTCQNRHDE, in        particular DNQ, notably DN,    -   substitution of the serine S in position 280 by a non-bulky        amino acid selected from the group consisting of GPLIVADCTN or        by a polar amino acid selected from the group consisting of        WYTCQNRKHDE, in particular GH,    -   substitution of the serine S in position 282 by a non-bulky        amino acid selected from the group consisting of GPLIVADCTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWYC, in particular ALV, notably AL,    -   and substitution of the isoleucine I in position 284 non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular VAL, notably V.

It means that at least one substitution among the 29 particularsubstitutions of set 7 in position G8, K9, V28, L47, K55, T69, L73, I77,V84, Y98, Y100, I123, N131, D142, N165, G194, R224, L227, L229, F230,L231, C259, 1262, W264, L275, A276, M281, L283 and T298 can beassociated with at least one substitution among the 21 particularsubstitutions of set 8 in position 1168, D192, Y258, C260, T261, D263,G265, I266, A267, K268, P269, E270, Y/L271, K272, P273, K274, P277,K278, S280, S282 and 1284.

The invention also relates to the above-mentioned mutatedhyperthermophilic PTE having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus acidocaldarius corresponding to thesequence SEQ ID NO: 5, and wherein the at least one mutation is selectedfrom the group consisting of:

-   -   substitution of the glycine G in position 8 by a non-bulky amino        acid selected from the group consisting of PLIVADCSTN, in        particular STA, notably S,    -   substitution of the lysine K in position 9 by a charged amino        acid selected from the group consisting of RHDEC, in particular        EDR, notably E,    -   substitution of the valine V in position 28 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular GIFA, notably A,    -   substitution of the leucine L in position 47 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular YW,    -   substitution of the lysine K in position 55 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular IRL, notably I,    -   substitution of the threonine T in position 69 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSN, in        particular VAS, notably S,    -   substitution of the leucine L in position 73 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular CAMI, notably I,    -   substitution of the isoleucine I in position 77 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular PTV, notably T,    -   substitution of the valine V in position 84 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular AGI, notably A,    -   substitution of the tyrosine Y in position 98 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular FCLW, notably W,    -   substitution of the tyrosine Y in position 100 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular GEWF, notably F,    -   substitution of the isoleucine I in position 123 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular LAV, notably L,    -   substitution of the asparagine N in position 131 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN, in        particular PST, notably P,    -   substitution of the aspartic acid D in position 142 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE, in        particular SET, notably T,    -   substitution of the asparagine N in position 165 by a polar        amino acid selected from the group consisting of WYSTCQRKHDE, in        particular QR,    -   substitution of the glycine G in position 194 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCNQRKHDE, in particular ST, notably S,    -   substitution of the arginine R in position 224 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNKHDE, in particular CSTAH, notably AC,    -   substitution of the leucine L in position 227 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by apolar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular AIVH, notably V,    -   substitution of the leucine L in position 229 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VIMFGAPWYC, in particular IM, notably M,    -   substitution of the phenylalanine F in position 230 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN, in particular LTAS, notably S,    -   substitution of the leucine L in position 231 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN, in        particular PVA, notably P,    -   substitution of the cysteine C in position 259 by a hydrophobic        amino acid selected from the group consisting of VILMFGAPWY, in        particular YLIA, notably LA,    -   substitution of the isoleucine I in position 262 by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWYC, in particular FWC, notably F,    -   substitution of the tryptophane W in position 264 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPYC, in particular ALMFCITV, notably F,    -   substitution of the leucine L in position 275 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular AVP, notably P,    -   substitution of the alanine A in position 276 by a hydrophobic        amino acid selected from the group consisting of VILMFGPWYC or        by a non-bulky amino acid selected from the group consisting of        GPLIVDCSTN, in particular NVMT, notably T,    -   substitution of the methionine M in position 281 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILFGAPWYC, in particular TYP, notably T,    -   substitution of the leucine L in position 283 by a bulky amino        acid selected from the group consisting of EKHRQYWFM, in        particular FMH, notably M,    -   and substitution of the threonine T in position 298 by a polar        amino acid selected from the group consisting of WYSCQNRKHDE, in        particular QS, notably S, further comprises at least one        mutation selected from the group consisting of:    -   substitution of the proline P in position 68 by a non-bulky        amino acid selected from the group consisting of GLIVADCSTN, in        particular GAV,    -   substitution of the glycine G in position 226 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN, in        particular AVP,    -   and substitution of the tryptophan W in position 279 by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPYC or by a charged amino acid selected from the group        consisting of RKHDEC, in particular RKDI, notably R.

It means that at least one substitution among the 29 particularsubstitutions of set 7 in position G8, K9, V28, L47, K55, T69, L73, I77,V84, Y98, Y100, I123, N131, D142, N165, G194, R224, L227, L229, F230,L231, C259, 1262, W264, L275, A276, M281, L283 and T298 can beassociated with at least one substitution among the 3 particularsubstitutions of set 9 in position P68, G226 and W279.

The invention also relates to the above-mentioned mutatedhyperthermophilic PTE having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus acidocaldarius corresponding to thesequence SEQ ID NO: 5, and wherein the at least one mutation selectedfrom the group consisting of:

-   -   substitution of the glycine G in position 8 by a non-bulky amino        acid selected from the group consisting of PLIVADCSTN, in        particular STA, notably S,    -   substitution of the lysine K in position 9 by a charged amino        acid selected from the group consisting of RHDEC, in particular        EDR, notably E,    -   substitution of the valine V in position 28 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular GIFA, notably A,    -   substitution of the leucine L in position 47 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular YW,    -   substitution of the lysine K in position 55 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular IRL, notably I,    -   substitution of the threonine T in position 69 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSN, in        particular VAS, notably S,    -   substitution of the leucine L in position 73 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular CAMI, notably I,    -   substitution of the isoleucine I in position 77 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular PTV, notably T,    -   substitution of the valine V in position 84 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular AGI, notably A,    -   substitution of the tyrosine Y in position 98 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular FCLW, notably W,    -   substitution of the tyrosine Y in position 100 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular GEWF, notably F,    -   substitution of the isoleucine I in position 123 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular LAV, notably L,    -   substitution of the asparagine N in position 131 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN, in        particular PST, notably P,    -   substitution of the aspartic acid D in position 142 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE, in        particular SET, notably T,    -   substitution of the asparagine N in position 165 by a polar        amino acid selected from the group consisting of WYSTCQRKHDE, in        particular QR,    -   substitution of the glycine G in position 194 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCNQRKHDE, in particular ST, notably S,    -   substitution of the arginine R in position 224 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNKHDE, in particular CSTAH, notably AC,    -   substitution of the leucine L in position 227 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by apolar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular AIVH, notably V,    -   substitution of the leucine L in position 229 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VIMFGAPWYC, in particular IM, notably M,    -   substitution of the phenylalanine F in position 230 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN, in particular LTAS, notably S,    -   substitution of the leucine L in position 231 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN, in        particular PVA, notably P,    -   substitution of the cysteine C in position 259 by a hydrophobic        amino acid selected from the group consisting of VILMFGAPWY, in        particular YLIA, notably LA,    -   substitution of the isoleucine I in position 262 by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWYC, in particular FWC, notably F,    -   substitution of the tryptophane W in position 264 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPYC, in particular ALMFCITV, notably F,    -   substitution of the leucine L in position 275 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular AVP, notably P,    -   substitution of the alanine A in position 276 by a hydrophobic        amino acid selected from the group consisting of VILMFGPWYC or        by a non-bulky amino acid selected from the group consisting of        GPLIVDCSTN, in particular NVMT, notably T,    -   substitution of the methionine M in position 281 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILFGAPWYC, in particular TYP, notably T,    -   substitution of the leucine L in position 283 by a bulky amino        acid selected from the group consisting of EKHRQYWFM, in        particular FMH, notably M,    -   and substitution of the threonine T in position 298 by a polar        amino acid selected from the group consisting of WYSCQNRKHDE, in        particular QS, notably S, further comprises at least one        mutation selected from the group consisting of:    -   substitution of the isoleucine I in position 168 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VLMFGAPWYC, in particular VAL, notably V,    -   substitution of the aspartic acid D in position 192 by a        non-bulky amino acid selected from the group consisting of        GPLVADCSTN or by a polar amino acid selected from the group        consisting of WYSTCQNRKHE, in particular ST, notably S,    -   substitution of the tyrosine Y position 258 by a non-bulky amino        acid selected from the group consisting of GPLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWC, in particular CSVW, notably C,    -   substitution of the cysteine C in position 260 a non-bulky amino        acid selected from the group consisting of GPLIVADSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWY, in particular SFWV, notably S,    -   substitution of the threonine T in position 261 a non-bulky        amino acid selected from the group consisting of GPLIVADCSN or        by a polar amino acid selected from the group consisting of        WYSCQNRKHE, in particular GH, notably G,    -   substitution of the aspartic acid D in position 263 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE or        by a non-bulky amino acid selected from the group consisting of        GPLIVACSTN, in particular SLH,    -   substitution of the glycine G in position 265 non-bulky amino        acid selected from the group consisting of PLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFAPWYC, in particular AVP,    -   substitution of the isoleucine I in position 266 by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWYC or by a non-bulky amino acid selected from the group        consisting of GPLVADCSTN, in particular VWP, notably V,    -   substitution of the alanine A in position 267 by a polar amino        acid selected from the group consisting of WYSTCQNRKHDE, in        particular NQ, notably N,    -   substitution of the lysine K in position 268 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWYC, in particular IAP, notably IP,    -   substitution of the proline P in position 269 by a hydrophobic        amino acid selected from the group consisting of VILMFGAWYC, in        particular MCL, notably M,    -   substitution of the glutamic acid E in position 270 by a polar        amino acid selected from the group consisting of WYSTCQNRKHD, in        particular DQ, notably D,    -   substitution of the tyrosine Y in position 271 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWC, in particular VAL,    -   substitution of the lysine K in position 272 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular MLA,    -   substitution of the proline P in position 273 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular DEL, notably DL,    -   substitution of the lysine K in position 274 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRHD, in particular RP,    -   substitution of the proline P in position 277 by a bulky amino        acid selected from the group consisting of EHKRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAWYC, in particular KAV, notably K,    -   substitution of the lysine K in position 278 by a polar amino        acid selected from the group consisting of WYSTCQNRHDE, in        particular DNQ, notably DN,    -   substitution of the serine S in position 280 by a non-bulky        amino acid selected from the group consisting of GPLIVADCTN or        by a polar amino acid selected from the group consisting of        WYTCQNRKHDE, in particular GH,    -   substitution of the serine S in position 282 by a non-bulky        amino acid selected from the group consisting of GPLIVADCTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWYC, in particular ALV, notably AL,    -   and substitution of the isoleucine I in position 284 non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular VAL, notably V, further comprises at least one        mutation selected from the group consisting of:    -   substitution of the proline P in position 68 by a non-bulky        amino acid selected from the group consisting of GLIVADCSTN, in        particular GAV,    -   substitution of the glycine G in position 226 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN, in        particular AVP,    -   and substitution of the tryptophan W in position 279 by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPYC or by a charged amino acid selected from the group        consisting of RKHDEC, in particular RKDI, notably R.

It means that at least one substitution among the 29 particularsubstitutions of set 7 in position G8, K9, V28, L47, K55, T69, L73, I77,V84, Y98, Y100, I123, N131, D142, N165, G194, R224, L227, L229, F230,L231, C259, 1262, W264, L275, A276, M281, L283 and T298 can beassociated with at least one substitution among the 21 particularsubstitutions of set 8 in position 1168, D192, Y258, C260, T261, D263,G265, I266, A267, K268, P269, E270, Y/L271, K272, P273, K274, P277,K278, S280, S282 and 1284 and with at least at least one substitutionamong the 3 particular substitutions of set 9 in position P68, G226 andW279.

The invention also relates to the above-mentioned mutatedhyperthermophilic PTE having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus acidocaldarius corresponding to thesequence SEQ ID NO: 5, and wherein the at least one mutation selectedfrom the group consisting of:

-   -   substitution of the isoleucine I in position 168 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VLMFGAPWYC, in particular VAL, notably V,    -   substitution of the aspartic acid D in position 192 by a        non-bulky amino acid selected from the group consisting of        GPLVADCSTN or by a polar amino acid selected from the group        consisting of WYSTCQNRKHE, in particular ST, notably S,    -   substitution of the tyrosine Y position 258 by a non-bulky amino        acid selected from the group consisting of GPLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWC, in particular CSVW, notably C,    -   substitution of the cysteine C in position 260 a non-bulky amino        acid selected from the group consisting of GPLIVADSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWY, in particular SFWV, notably S,    -   substitution of the threonine T in position 261 a non-bulky        amino acid selected from the group consisting of GPLIVADCSN or        by a polar amino acid selected from the group consisting of        WYSCQNRKHE, in particular GH, notably G,    -   substitution of the aspartic acid D in position 263 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE or        by a non-bulky amino acid selected from the group consisting of        GPLIVACSTN, in particular SLH,    -   substitution of the glycine G in position 265 non-bulky amino        acid selected from the group consisting of PLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFAPWYC, in particular AVP,    -   substitution of the isoleucine I in position 266 by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWYC or by a non-bulky amino acid selected from the group        consisting of GPLVADCSTN, in particular VWP, notably V,    -   substitution of the alanine A in position 267 by a polar amino        acid selected from the group consisting of WYSTCQNRKHDE, in        particular NQ, notably N,    -   substitution of the lysine K in position 268 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWYC, in particular IAP, notably IP,    -   substitution of the proline P in position 269 by a hydrophobic        amino acid selected from the group consisting of VILMFGAWYC, in        particular MCL, notably M,    -   substitution of the glutamic acid E in position 270 by a polar        amino acid selected from the group consisting of WYSTCQNRKHD, in        particular DQ, notably D,    -   substitution of the tyrosine Y in position 271 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWC, in particular VAL,    -   substitution of the lysine K in position 272 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular MLA,    -   substitution of the proline P in position 273 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular DEL, notably DL,    -   substitution of the lysine K in position 274 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRHD, in particular RP,    -   substitution of the proline P in position 277 by a bulky amino        acid selected from the group consisting of EHKRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAWYC, in particular KAV, notably K,    -   substitution of the lysine K in position 278 by a polar amino        acid selected from the group consisting of WYSTCQNRHDE, in        particular DNQ, notably DN,    -   substitution of the serine S in position 280 by a non-bulky        amino acid selected from the group consisting of GPLIVADCTN or        by a polar amino acid selected from the group consisting of        WYTCQNRKHDE, in particular GH,    -   substitution of the serine S in position 282 by a non-bulky        amino acid selected from the group consisting of GPLIVADCTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWYC, in particular ALV, notably AL,    -   and substitution of the isoleucine I in position 284 non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular VAL, notably V, further comprises at least one        mutation selected from the group consisting of:    -   substitution of the proline P in position 68 by a non-bulky        amino acid selected from the group consisting of GLIVADCSTN, in        particular GAV,    -   substitution of the glycine G in position 226 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN, in        particular AVP,    -   and substitution of the tryptophan W in position 279 by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPYC or by a charged amino acid selected from the group        consisting of RKHDEC, in particular RKDI, notably R.

It means that at least one substitution among the 21 particularsubstitutions of set 8 in position 1168, D192, Y258, C260, T261, D263,G265, I266, A267, K268, P269, E270, Y/L271, K272, P273, K274, P277,K278, S280, S282 and 1284 can be associated with at least at least onesubstitution among the 3 particular substitutions of set 9 in positionP68, G226 and W279.

A more particular subject of the invention is mutated hyperthermophilicPTE having a lactonase activity according to the present invention,derived from the hyperthermophilic PTE of Sulfolobus acidocaldariuscorresponding to the sequence SEQ ID NO: 5, said mutatedhyperthermophilic PTE correspond to the following sequences:

-   -   SEQ ID NO: 51 corresponding to the SEQ ID NO: 5 comprising the        following one mutation: substitution of the tryptophan W in        position 264 by a phenylalanine F,    -   SEQ ID NO: 53 corresponding to the SEQ ID NO: 5 comprising the        following one mutation: substitution of the tryptophan W in        position 264 by a methionine M,    -   SEQ ID NO: 55 corresponding to the SEQ ID NO: 5 comprising the        following one mutation: substitution of the tryptophan W in        position 264 by a leucine L,    -   SEQ ID NO: 57 corresponding to the SEQ ID NO: 5 comprising the        following one mutation: substitution of the tryptophan W in        position 264 by an alanine A,    -   SEQ ID NO: 59 corresponding to the SEQ ID NO: 5 comprising the        following one mutation: substitution of the tryptophan W in        position 264 by an isoleucine I,    -   SEQ ID NO: 61 corresponding to the SEQ ID NO: 5 comprising the        following one mutation: substitution of the tryptophan W in        position 264 by a valine V,    -   SEQ ID NO: 63 corresponding to the SEQ ID NO: 5 comprising the        following one mutation: substitution of the tryptophan W in        position 264 by a threonine T,    -   SEQ ID NO: 65 corresponding to the SEQ ID NO: 5 comprising the        following three mutations: substitution of the cysteine C in        position 259 by a leucine L, substitution of the isoleucine I in        position 262 by a phenylalanine F, substitution of the        tryptophan W in position 264 by an alanine A,    -   SEQ ID NO: 67 corresponding to the SEQ ID NO: 5 comprising the        following four mutations: substitution of the valine V in        position 28 by an alanine A, substitution of the tyrosine Y in        position 98 by a tryptophan W, substitution of the leucine L in        position 229 by a methionine M, substitution of the tryptophan W        in position 264 by a methionine M,    -   SEQ ID NO: 69 corresponding to the SEQ ID NO: 5 comprising the        following four mutations: substitution of the valine V in        position 28 by an alanine A, substitution of the tyrosine Y in        position 98 by a tryptophan W, substitution of the tryptophan W        in position 264 by a leucine L, substitution of the methionine M        in position 281 by a threonine T,    -   SEQ ID NO: 71 corresponding to the SEQ ID NO: 5 comprising the        following four mutations: substitution of the cytosine C in        position 259 by an alanine A, substitution of the tryptophan W        in position 264 by a methionine M, substitution of the        methionine M in position 281 by a threonine T,    -   SEQ ID NO: 73 corresponding to the SEQ ID NO: 5 comprising the        following six mutations: substitution of the valine V in        position 28 by an alanine A, substitution of the isoleucine I in        position 77 by a threonine T, substitution of the tyrosine Y in        position 98 by a tryptophan W, substitution of the tyrosine Y in        position 100 by a phenylalanine F, substitution of the        asparagine N in position 131 by a proline P, substitution of the        leucine L in position 227 by a valine V,    -   SEQ ID NO: 75 corresponding to the SEQ ID NO: 5 comprising the        following six mutations: substitution of the leucine L in        position 73 by an isoleucine I, substitution of the tyrosine Y        in position 100 by a phenylalanine F, substitution of the        isoleucine I in position 123 by a leucine L, substitution of the        leucine L in position 229 by a methionine M, substitution of the        phenylalanine F in position 230 by a serine S, substitution of        the tryptophan W in position 264 by a leucine L,    -   SEQ ID NO: 77 corresponding to the SEQ ID NO: 5 comprising the        following seven mutations: substitution of the threonine T in        position 69 by a serine S, substitution of the leucine L in        position 73 by an isoleucine I, substitution of the asparagine N        in position 131 by a proline P, substitution of the leucine L in        position 229 by a methionine M, substitution of the        phenylalanine F in position 230 by a serine S, substitution of        the tryptophan W in position 264 by a methionine M, substitution        of the leucine L in position 275 by a proline P,    -   SEQ ID NO: 79 corresponding to the SEQ ID NO: 5 comprising the        following six mutations: substitution of the threonine T in        position 69 by a serine S, substitution of the isoleucine I in        position 77 by a threonine T, substitution of the tyrosine Y in        position 98 by a tryptophan W, substitution of the tyrosine Y in        position 100 by a phenylalanine F, substitution of the leucine L        in position 229 by a methionine M, substitution of the        tryptophan W in position 264 by a leucine L,    -   SEQ ID NO: 81 corresponding to the SEQ ID NO: 5 comprising the        following five mutations: substitution of the lysine K in        position 9 by a glutamic acid E, substitution of the tyrosine Y        in position 98 by a tryptophan W, substitution of the tyrosine Y        in position 100 by a phenylalanine F, substitution of the        leucine L in position 229 by a methionine M,    -   SEQ ID NO: 83 corresponding to the SEQ ID NO: 5 comprising the        following two mutations: substitution of the leucine L in        position 73 by an isoleucine I, substitution of the tryptophan W        in position 264 by a phenylalanine F,    -   SEQ ID NO: 85 corresponding to the SEQ ID NO: 5 comprising the        following five mutations: substitution of the threonine T in        position 69 by a serine S, substitution of the tyrosine Y in        position 98 by a tryptophan W, substitution of the tyrosine Y in        position 100 by a phenylalanine F, substitution of the        asparagine N in position 131 by a proline P, substitution of the        leucine L in position 229 by a methionine M,    -   SEQ ID NO: 87 corresponding to the SEQ ID NO: 5 comprising the        following four mutations: substitution of the valine V in        position 28 by an alanine A, substitution of the leucine L in        position 227 by a valine V, substitution the tryptophan W in        position 264 by a leucine L,    -   SEQ ID NO: 89 corresponding to the SEQ ID NO: 5 comprising the        following eight mutations: substitution of the proline P in        position 68 by a valine V, substitution of the threonine T in        position 69 by a serine S, substitution of the tyrosine Y in        position 98 by a tryptophan W, substitution of the tyrosine Y in        position 100 by a phenylalanine F, substitution of the leucine L        in position 229 by a methionine M, substitution of the cysteine        C in position 259 by an alanine A, substitution the tryptophan W        in position 264 by a leucine L, substitution of the methionine M        in position 281 by a threonine T,    -   SEQ ID NO: 91 corresponding to the SEQ ID NO: 5 comprising the        following eight mutations: substitution of the threonine T in        position 69 by a serine S, substitution of the tyrosine Y in        position 98 by a tryptophan W, substitution of the tyrosine Y in        position 100 by a phenylalanine F, substitution of the        asparagine N in position 131 by a proline P, substitution of the        leucine L in position 227 by a valine V, substitution the        tryptophan W in position 264 by a methionine M,    -   SEQ ID NO: 93 corresponding to the SEQ ID NO: 5 comprising the        following five mutations: substitution of the threonine T in        position 69 by a serine S, substitution of the leucine L in        position 73 by an isoleucine I, substitution of the tyrosine Y        in position 98 by a tryptophan W, substitution of the tyrosine Y        in position 100 by a phenylalanine F, substitution of the        asparagine N in position 131 by a proline P.

The coding sequence of the above-mentioned mutated hyperthermophilic PTEhaving a lactonase activity according to the present invention, derivedfrom the hyperthermophilic PTE of Sulfolobus acidocaldariuscorresponding to the sequence SEQ ID NO: 5 and corresponding to thefollowing sequences SEQ ID NO: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90 and 92 are also part of theinvention.

The invention also related to mutated hyperthermophilic PTE having alactonase activity according to the present invention, derived from thehyperthermophilic PTE of Sulfolobus acidocaldarius corresponding to thesequence SEQ ID NO: 5, said mutated hyperthermophilic PTE correspond tothe following sequences SEQ ID NO: 181, 183, 185, 187, 189, 191, 193,195, 197, 199, 201, 203, 205, 207, 209, 21, 213, 215, 217, 219, 221 and223 for the proteins and to their respective coding sequences SEQ ID NO:180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206,208, 210, 212, 214, 216, 218, 220 and 222.

The invention relates more particularly to the above-mentioned mutatedhyperthermophilic PTE having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus acidocaldarius corresponding to thesequence SEQ ID NO: 5, further comprising at least one mutationcorresponding to a substitution of at least one of the amino acids ofthe following amino acid pairs, the positions of which in SEQ ID NO: 5are indicated hereafter, by another natural or non-natural amino acid:3K/315S, 15G/13S, 27R/76D, 27R/43E, 34R/43E, 34R/46E, 56T/53E, 56T/286T,75R/122D, 82K/43E, 82K/44D, 85K/81E, 110R/114E, 124K/163E, 148R/149D,152R/149D, 155R/151E, 155R/188E, 155R/189E, 162K/189E, 184R/151E,184R/188E, 184R/181E, 211K/246D, 216K/215D, 224R/257D, 224R/203D,235K/205D, 236R/203D, 242K/246D, 246D/245K, 251R/250D, 278K/287D,293K/299D, 311A/308K.

The invention relates also to the above-mentioned mutatedhyperthermophilic PTEs having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus islandicus corresponding to thesequence SEQ ID NO: 7, said mutated PTEs comprise the at least onemutation selected from the group consisting of: substitutions of theglycine G in position 7, the lysine K in position 8, the valine V inposition 27, the leucine L in position 46, the lysine K in position 54,the proline P in position 67, the threonine T in position 68, theleucine L in position 72, the isoleucine I in position 76, the valine Vin position 83, the tyrosine Y in position 97, the tyrosine Y inposition 99, the isoleucine I in position 122, the serine S in position130, the aspartic acid D in position 141, the lysine K in position 164,the isoleucine I in position 167, the aspartic acid D in position 191,the glycine G in position 193, the arginine R in position 223, theglycine G in position 225, the leucine L in position 226, the leucine Lin position 228, the phenylalanine F in position 229, the leucine L inposition 230, the tyrosine Y position 257, the cysteine C in position258, the cysteine C in position 259, the threonine T in position 260,the isoleucine I in position 261, the aspartic acid D in position 262,the tryptophane W in position 263, the glycine G in position 264, thethreonine T in position 265, the alanine A in position 266, the arginineR in position 267, the proline P in position 268, the glutamic acid E inposition 269, the leucine L in position 270, the lysine K in position271, the proline P in position 272, the lysine K in position 273, theleucine L in position 274, the alanine A in position 275, the proline Pin position 276, the arginine R in position 277, the tryptophan W inposition 278, the serine S in position 279, the methionine M in position280, the alanine A in position 281, the serine S in position 282, theisoleucine I in position 283 and the serine S in position 297, of SEQ IDNO: 7 by any other natural or non-natural amino acid.

In a more specific embodiment, the above-mentioned mutatedhyperthermophilic PTEs having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus islandicus corresponding to thesequence SEQ ID NO: 7, comprise the at least one mutation selected fromthe group consisting of: substitutions of the valine V in position 27,the proline P in position 67, the threonine T in position 68, theleucine L in position 72, the tyrosine Y in position 97, the tyrosine Yin position 99, the aspartic acid D in position 141, the arginine R inposition 223, the glycine G in position 225, the leucine L in position226, the phenylalanine F in position 229, the cysteine C in position258, the tryptophane W in position 263 and the tryptophan W in position278, of SEQ ID NO: 7 by any other natural or non-natural amino acid.

In an even more specific embodiment, the above-mentioned mutatedhyperthermophilic PTEs having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus islandicus corresponding to thesequence SEQ ID NO: 7, comprise only one mutation, said mutation beingselected from the group consisting of: substitutions of the valine V inposition 27, the proline P in position 67, the threonine T in position68, the leucine L in position 72, the aspartic acid D in position 141,the glycine G in position 225, the leucine L in position 226, thephenylalanine F in position 229, the tryptophane W in position 263 andthe tryptophan W in position 278, of SEQ ID NO: 7 by any other naturalor non-natural amino acid.

In an even more specific embodiment, the above-mentioned mutatedhyperthermophilic PTEs having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus islandicus corresponding to thesequence SEQ ID NO: 7, comprise the at least one mutation selected fromthe group consisting of: substitutions of the glycine G in position 7,the lysine K in position 8, the leucine L in position 46, the lysine Kin position 54, the isoleucine I in position 76, the valine V inposition 83, the isoleucine I in position 122, the serine S in position130, the lysine K in position 164, the isoleucine I in position 167, theaspartic acid D in position 191, the glycine G in position 193, theleucine L in position 228, the leucine L in position 230, the tyrosine Yposition 257, the cysteine C in position 259, the threonine T inposition 260, the isoleucine I in position 261, the aspartic acid D inposition 262, the glycine G in position 264, the threonine T in position265, the alanine A in position 266, the arginine R in position 267, theproline P in position 268, the glutamic acid E in position 269, theleucine L in position 270, the lysine K in position 271, the proline Pin position 272, the lysine K in position 273, the leucine L in position274, the alanine A in position 275, the proline P in position 276, thearginine R in position 277, the serine S in position 279, the methionineM in position 280, the alanine A in position 281, the leucine L inposition 282, the isoleucine I in position 283 and the serine S inposition 297, of SEQ ID NO: 7 by any other natural or non-natural aminoacid.

In an even more specific embodiment, the above-mentioned mutatedhyperthermophilic PTEs having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus islandicus corresponding to thesequence SEQ ID NO: 7, comprise only one mutation, said mutation beingselected from the group consisting of: substitutions of the glycine G inposition 7, the lysine K in position 8, the leucine L in position 46,the lysine K in position 54, the isoleucine I in position 76, the valineV in position 83, the isoleucine I in position 122, the serine S inposition 130, the lysine K in position 164, the isoleucine I in position167, the aspartic acid D in position 191, the glycine G in position 193,the leucine L in position 228, the leucine L in position 230, thetyrosine Y position 257, the cysteine C in position 259, the threonine Tin position 260, the isoleucine I in position 261, the aspartic acid Din position 262, the glycine G in position 264, the threonine T inposition 265, the alanine A in position 266, the arginine R in position267, the proline P in position 268, the glutamic acid E in position 269,the leucine L in position 270, the lysine K in position 271, the prolineP in position 272, the lysine K in position 273, the leucine L inposition 274, the alanine A in position 275, the proline P in position276, the arginine R in position 277, the serine S in position 279, themethionine M in position 280, the alanine A in position 281, the leucineL in position 282, the isoleucine I in position 283, the serine S inposition 297, of SEQ ID NO: 7 by any other natural or non-natural aminoacid.

The invention relates more particularly to the above-mentioned mutatedhyperthermophilic PTE having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus islandicus corresponding to thesequence SEQ ID NO: 7, and wherein the at least one mutation is selectedfrom the group consisting of:

-   -   substitution of the glycine G in position 7 by a non-bulky amino        acid selected from the group consisting of PLIVADCSTN, in        particular STA, notably S,    -   substitution of the lysine K in position 8 by a charged amino        acid selected from the group consisting of RHDEC, in particular        EDR, notably E,    -   substitution of the valine V in position 27 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular GIFA, notably A,    -   substitution of the leucine L in position 46 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular YW,    -   substitution of the lysine K in position 54 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular IRL, notably I,    -   substitution of the threonine T in position 68 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSN, in        particular VAS, notably S,    -   substitution of the leucine L in position 72 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular CAMI, notably I,    -   substitution of the isoleucine I in position 76 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular PTV, notably T,    -   substitution of the valine V in position 83 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular AGI, notably A,    -   substitution of the tyrosine Y in position 97 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular FCLW, notably W,    -   substitution of the tyrosine Y in position 99 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular GEWF, notably F,    -   substitution of the isoleucine I in position 122 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular LAV, notably L,    -   substitution of the serine S in position 130 by a non-bulky        amino acid selected from the group consisting of GPLIVADCTN, in        particular PT, notably P,    -   substitution of the aspartic acid D in position 141 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE, in        particular SET, notably T,    -   substitution of the lysine K in position 164 by a polar amino        acid selected from the group consisting of WYSTCQNRHDE, in        particular NQR, notably N,    -   substitution of the glycine G in position 193 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCNQRKHDE, in particular ST, notably S,    -   substitution of the arginine R in position 223 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNKHDE, in particular CSTAH, notably AC,    -   substitution of the leucine L in position 226 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by apolar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular AIVH, notably V,    -   substitution of the leucine L in position 228 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VIMFGAPWYC, in particular IM, notably M,    -   substitution of the phenylalanine F in position 229 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN, in particular LTAS, notably S,    -   substitution of the leucine L in position 230 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN, in        particular PVA, notably P,    -   substitution of the cysteine C in position 258 by a hydrophobic        amino acid selected from the group consisting of VILMFGAPWY, in        particular YLIA, notably LA,    -   substitution of the isoleucine I in position 261 by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWYC, in particular FWC, notably F,    -   substitution of the tryptophane W in position 263 by a non-bulky        amino acid selected from the group consisting of GPLIVDCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPYC, in particular ALMFCITV, notably F,    -   substitution of the leucine L in position 274 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular AVP, notably P,    -   substitution of the alanine A in position 275 by a hydrophobic        amino acid selected from the group consisting of VILMFGPWYC or        by a non-bulky amino acid selected from the group consisting of        GPLIVDCSTN, in particular NVMT, notably T,    -   substitution of the methionine M in position 280 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VLMFGAPWYC, in particular TYP, notably T,    -   substitution of the leucine L in position 282 by a bulky amino        acid selected from the group consisting of EKHRQYWFM, in        particular FMH, notably M,    -   and substitution of the serine S in position 297 by a polar        amino acid selected from the group consisting of WYTCQNRKHDE, in        particular QT.

These 29 particular substitutions in position G7, K8, V27, F46, K54,T68, L72, I76, V83, Y97, Y99, I122, S130, D141, K164, G193, R223, L226,L228, F229, L230, C258, 1261, W263, L274, A275, M280, L282 and N297belong to the tenth set of substitutions called set 10.

The invention relates more particularly to the above-mentioned mutatedhyperthermophilic PTE having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus islandicus corresponding to thesequence SEQ ID NO: 7, and wherein the at least one mutation is selectedfrom the group consisting of:

-   -   substitution of the isoleucine I in position 167 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VLMFGAPWYC, in particular VAL, notably V,    -   substitution of the aspartic acid D in position 191 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN or by a polar amino acid selected from the group        consisting of WYSTCQNRKHE, in particular ST, notably S,    -   substitution of the tyrosine Y position 257 by a non-bulky amino        acid selected from the group consisting of GPLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWC, in particular CSVW, notably C,    -   substitution of the cysteine C in position 259 a non-bulky amino        acid selected from the group consisting of GPLIVADSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWY, in particular SFWV, notably S,    -   substitution of the threonine T in position 260 a non-bulky        amino acid selected from the group consisting of GPLIVADCSN or        by a polar amino acid selected from the group consisting of        WYSCQNRKHE, in particular GH, notably G,    -   substitution of the aspartic acid D in position 262 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE or        by a non-bulky amino acid selected from the group consisting of        GPLIVADCSTN, in particular SLH,    -   substitution of the glycine G in position 264 non-bulky amino        acid selected from the group consisting of PLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFAPWYC, in particular AVP,    -   substitution of the threonine T in position 265 by a hydrophobic        amino acid selected from the group consisting of VILMFGAPWYC or        by a non-bulky amino acid selected from the group consisting of        GPLIVADCSN, in particular VWP, notably V,    -   substitution of the alanine A in position 266 by a polar amino        acid selected from the group consisting of WYSTCQNRKHDE, in        particular NQ, notably N,    -   substitution of the arginine R in position 267 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWYC, in particular IAP, notably IP,    -   substitution of the proline P in position 268 by a hydrophobic        amino acid selected from the group consisting of VILMFGAWYC, in        particular MCL, notably M,    -   substitution of the glutamic acid E in position 269 by a polar        amino acid selected from the group consisting of WYSTCQNRKHD, in        particular DQ, notably D,    -   substitution of the leucine L in position 270 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWC, in particular VA,    -   substitution of the lysine K in position 271 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular MLA,    -   substitution of the proline P in position 272 by non-bulky amino        acid selected from the group consisting of GPLIVADCSTN or by a        polar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular DEL, notably DL,    -   substitution of the lysine K in position 273 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRHD, in particular RP,    -   substitution of the proline P in position 276 by a bulky amino        acid selected from the group consisting of EHKRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAWYC, in particular KAV, notably K,    -   substitution of the arginine R in position 277 by a polar amino        acid selected from the group consisting of WYSTCQNKHDE, in        particular DNQ, notably DN,    -   substitution of the serine S in position 279 by a non-bulky        amino acid selected from the group consisting of GPLIVADCTN or        by a polar amino acid selected from the group consisting of        WYTCQNRKHDE, in particular GH,    -   substitution of the alanine A in position 281 by a non-bulky        amino acid selected from the group consisting of GPLIVDCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWYC, in particular ALV, notably AL,    -   and substitution of the isoleucine I in position 283 non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular VAL, notably V.

These 21 particular substitutions in position 1167, D191, Y257, C259,T260, D262, G264, T265, A266, R267, P268, E269, L270, K271, P272, K273,P276, R277, S279, A281 and I283 belong to the eleventh set ofsubstitutions called set 11.

The invention relates more particularly to the above-mentioned mutatedhyperthermophilic PTE having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus islandicus corresponding to thesequence SEQ ID NO: 7, and wherein the at least one mutation is selectedfrom the group consisting of:

-   -   substitution of the proline P in position 67 by a non-bulky        amino acid selected from the group consisting of GLIVADCSTN, in        particular GAV,    -   substitution of the glycine G in position 225 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN, in        particular AVP,    -   and substitution of the tryptophan W in position 278 by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPYC or by a charged amino acid selected from the group        consisting of RKHDEC, in particular RKDI, notably R.

These 3 particular substitutions in position P67, G225 and W278 belongto the tlewth set of substitutions called set 12.

The invention also relates to the above-mentioned mutatedhyperthermophilic PTE having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus islandicus corresponding to thesequence SEQ ID NO: 7, wherein the at least one mutation selected fromthe group consisting of:

-   -   substitution of the glycine G in position 7 by a non-bulky amino        acid selected from the group consisting of PLIVADCSTN, in        particular STA, notably S,    -   substitution of the lysine K in position 8 by a charged amino        acid selected from the group consisting of RHDEC, in particular        EDR, notably E,    -   substitution of the valine V in position 27 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular GIFA, notably A,    -   substitution of the leucine L in position 46 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular YW,    -   substitution of the lysine K in position 54 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular IRL, notably I,    -   substitution of the threonine T in position 68 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSN, in        particular VAS, notably S,    -   substitution of the leucine L in position 72 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular CAMI, notably I,    -   substitution of the isoleucine I in position 76 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular PTV, notably T,    -   substitution of the valine V in position 83 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular AGI, notably A,    -   substitution of the tyrosine Y in position 97 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular FCLW, notably W,    -   substitution of the tyrosine Y in position 99 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular GEWF, notably F,    -   substitution of the isoleucine I in position 122 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular LAV, notably L,    -   substitution of the serine S in position 130 by a non-bulky        amino acid selected from the group consisting of GPLIVADCTN, in        particular PT, notably P,    -   substitution of the aspartic acid D in position 141 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE, in        particular SET, notably T,    -   substitution of the lysine K in position 164 by a polar amino        acid selected from the group consisting of WYSTCQNRHDE, in        particular NQR, notably N,    -   substitution of the glycine G in position 193 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCNQRKHDE, in particular ST, notably S,    -   substitution of the arginine R in position 223 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNKHDE, in particular CSTAH, notably AC,    -   substitution of the leucine L in position 226 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by apolar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular AIVH, notably V,    -   substitution of the leucine L in position 228 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VIMFGAPWYC, in particular IM, notably M,    -   substitution of the phenylalanine F in position 229 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN, in particular LTAS, notably S,    -   substitution of the leucine L in position 230 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN, in        particular PVA, notably P,    -   substitution of the cysteine C in position 258 by a hydrophobic        amino acid selected from the group consisting of VILMFGAPWY, in        particular YLIA, notably LA,    -   substitution of the isoleucine I in position 261 by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWYC, in particular FWC, notably F,    -   substitution of the tryptophane W in position 263 by a non-bulky        amino acid selected from the group consisting of GPLIVDCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPYC, in particular ALMFCITV, notably F,    -   substitution of the leucine L in position 274 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular AVP, notably P,    -   substitution of the alanine A in position 275 by a hydrophobic        amino acid selected from the group consisting of VILMFGPWYC or        by a non-bulky amino acid selected from the group consisting of        GPLIVDCSTN, in particular NVMT, notably T,    -   substitution of the methionine M in position 280 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VLMFGAPWYC, in particular TYP, notably T,    -   substitution of the leucine L in position 282 by a bulky amino        acid selected from the group consisting of EKHRQYWFM, in        particular FMH, notably M,    -   and substitution of the serine S in position 297 by a polar        amino acid selected from the group consisting of WYTCQNRKHDE, in        particular QT, further comprises at least one mutation selected        from the group consisting of:    -   substitution of the isoleucine I in position 167 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VLMFGAPWYC, in particular VAL, notably V,    -   substitution of the aspartic acid D in position 191 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN or by a polar amino acid selected from the group        consisting of WYSTCQNRKHE, in particular ST, notably S,    -   substitution of the tyrosine Y position 257 by a non-bulky amino        acid selected from the group consisting of GPLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWC, in particular CSVW, notably C,    -   substitution of the cysteine C in position 259 a non-bulky amino        acid selected from the group consisting of GPLIVADSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWY, in particular SFWV, notably S,    -   substitution of the threonine T in position 260 a non-bulky        amino acid selected from the group consisting of GPLIVADCSN or        by a polar amino acid selected from the group consisting of        WYSCQNRKHE, in particular GH, notably G,    -   substitution of the aspartic acid D in position 262 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE or        by a non-bulky amino acid selected from the group consisting of        GPLIVADCSTN, in particular SLH,    -   substitution of the glycine G in position 264 non-bulky amino        acid selected from the group consisting of PLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFAPWYC, in particular AVP,    -   substitution of the threonine T in position 265 by a hydrophobic        amino acid selected from the group consisting of VILMFGAPWYC or        by a non-bulky amino acid selected from the group consisting of        GPLIVADCSN, in particular VWP, notably V,    -   substitution of the alanine A in position 266 by a polar amino        acid selected from the group consisting of WYSTCQNRKHDE, in        particular NQ, notably N,    -   substitution of the arginine R in position 267 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWYC, in particular IAP, notably IP,    -   substitution of the proline P in position 268 by a hydrophobic        amino acid selected from the group consisting of VILMFGAWYC, in        particular MCL, notably M,    -   substitution of the glutamic acid E in position 269 by a polar        amino acid selected from the group consisting of WYSTCQNRKHD, in        particular DQ, notably D,    -   substitution of the leucine L in position 270 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWC, in particular VA,    -   substitution of the lysine K in position 271 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular MLA,    -   substitution of the proline P in position 272 by non-bulky amino        acid selected from the group consisting of GPLIVADCSTN or by a        polar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular DEL, notably DL,    -   substitution of the lysine K in position 273 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRHD, in particular RP,    -   substitution of the proline P in position 276 by a bulky amino        acid selected from the group consisting of EHKRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAWYC, in particular KAV, notably K,    -   substitution of the arginine R in position 277 by a polar amino        acid selected from the group consisting of WYSTCQNKHDE, in        particular DNQ, notably DN,    -   substitution of the serine S in position 279 by a non-bulky        amino acid selected from the group consisting of GPLIVADCTN or        by a polar amino acid selected from the group consisting of        WYTCQNRKHDE, in particular GH,    -   substitution of the alanine A in position 281 by a non-bulky        amino acid selected from the group consisting of GPLIVDCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWYC, in particular ALV, notably AL,    -   and substitution of the isoleucine I in position 283 non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular VAL, notably V.

It means that at least one substitution among the 29 particularsubstitutions of set 10 in position G7, K8, V27, F46, K54, T68, L72,176, V83, Y97, Y99, I122, S130, D141, K164, G193, R223, L226, L228,F229, L230, C258, 1261, W263, L274, A275, M280, L282 and N297 can beassociated with at least one substitution among the 21 particularsubstitutions of set 11 in position 1167, D191, Y257, C259, T260, D262,G264, T265, A266, R267, P268, E269, L270, K271, P272, K273, P276, R277,S279, A281 and 1283.

The invention also relates to the above-mentioned mutatedhyperthermophilic PTE having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus islandicus corresponding to thesequence SEQ ID NO: 7, wherein the at least one mutation selected fromthe group consisting of:

-   -   substitution of the glycine G in position 7 by a non-bulky amino        acid selected from the group consisting of PLIVADCSTN, in        particular STA, notably S,    -   substitution of the lysine K in position 8 by a charged amino        acid selected from the group consisting of RHDEC, in particular        EDR, notably E,    -   substitution of the valine V in position 27 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular GIFA, notably A,    -   substitution of the leucine L in position 46 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular YW,    -   substitution of the lysine K in position 54 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular IRL, notably I,    -   substitution of the threonine T in position 68 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSN, in        particular VAS, notably S,    -   substitution of the leucine L in position 72 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular CAMI, notably I,    -   substitution of the isoleucine I in position 76 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular PTV, notably T,    -   substitution of the valine V in position 83 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular AGI, notably A,    -   substitution of the tyrosine Y in position 97 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular FCLW, notably W,    -   substitution of the tyrosine Y in position 99 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular GEWF, notably F,    -   substitution of the isoleucine I in position 122 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular LAV, notably L,    -   substitution of the serine S in position 130 by a non-bulky        amino acid selected from the group consisting of GPLIVADCTN, in        particular PT, notably P,    -   substitution of the aspartic acid D in position 141 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE, in        particular SET, notably T,    -   substitution of the lysine K in position 164 by a polar amino        acid selected from the group consisting of WYSTCQNRHDE, in        particular NQR, notably N,    -   substitution of the glycine G in position 193 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCNQRKHDE, in particular ST, notably S,    -   substitution of the arginine R in position 223 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNKHDE, in particular CSTAH, notably AC,    -   substitution of the leucine L in position 226 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by apolar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular AIVH, notably V,    -   substitution of the leucine L in position 228 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VIMFGAPWYC, in particular IM, notably M,    -   substitution of the phenylalanine F in position 229 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN, in particular LTAS, notably S,    -   substitution of the leucine L in position 230 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN, in        particular PVA, notably P,    -   substitution of the cysteine C in position 258 by a hydrophobic        amino acid selected from the group consisting of VILMFGAPWY, in        particular YLIA, notably LA,    -   substitution of the isoleucine I in position 261 by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWYC, in particular FWC, notably F,    -   substitution of the tryptophane W in position 263 by a non-bulky        amino acid selected from the group consisting of GPLIVDCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPYC, in particular ALMFCITV, notably F,    -   substitution of the leucine L in position 274 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular AVP, notably P,    -   substitution of the alanine A in position 275 by a hydrophobic        amino acid selected from the group consisting of VILMFGPWYC or        by a non-bulky amino acid selected from the group consisting of        GPLIVDCSTN, in particular NVMT, notably T,    -   substitution of the methionine M in position 280 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VLMFGAPWYC, in particular TYP, notably T,    -   substitution of the leucine L in position 282 by a bulky amino        acid selected from the group consisting of EKHRQYWFM, in        particular FMH, notably M,    -   and substitution of the serine S in position 297 by a polar        amino acid selected from the group consisting of WYTCQNRKHDE, in        particular QT, further comprises at least one mutation selected        from the group consisting of:    -   substitution of the proline P in position 67 by a non-bulky        amino acid selected from the group consisting of GLIVADCSTN, in        particular GAV,    -   substitution of the glycine G in position 225 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN, in        particular AVP,    -   and substitution of the tryptophan W in position 278 by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPYC or by a charged amino acid selected from the group        consisting of RKHDEC, in particular RKDI, notably R.

It means that at least one substitution among the 29 particularsubstitutions of set 10 in position G7, K8, V27, F46, K54, T68, L72,176, V83, Y97, Y99, I122, S130, D141, K164, G193, R223, L226, L228,F229, L230, C258, 1261, W263, L274, A275, M280, L282 and N297 can beassociated with at least one substitution among the, K273, P276, R277,S279, A281 and 1283 and with at least one substitution among the 3particular substitutions of set 12 in position P67, G225 and W278.

The invention also relates to the above-mentioned mutatedhyperthermophilic PTE having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus islandicus corresponding to thesequence SEQ ID NO: 7, wherein the at least one mutation selected fromthe group consisting of:

-   -   substitution of the glycine G in position 7 by a non-bulky amino        acid selected from the group consisting of PLIVADCSTN, in        particular STA, notably S,    -   substitution of the lysine K in position 8 by a charged amino        acid selected from the group consisting of RHDEC, in particular        EDR, notably E,    -   substitution of the valine V in position 27 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular GIFA, notably A,    -   substitution of the leucine L in position 46 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular YW,    -   substitution of the lysine K in position 54 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular IRL, notably I,    -   substitution of the threonine T in position 68 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSN, in        particular VAS, notably S,    -   substitution of the leucine L in position 72 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular CAMI, notably I,    -   substitution of the isoleucine I in position 76 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular PTV, notably T,    -   substitution of the valine V in position 83 by a non-bulky amino        acid selected from the group consisting of GPLIADCSTN or by a        hydrophobic amino acid selected from the group consisting of        ILMFGAPWYC, in particular AGI, notably A,    -   substitution of the tyrosine Y in position 97 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular FCLW, notably W,    -   substitution of the tyrosine Y in position 99 by a bulky amino        acid selected from the group consisting of EHKRQWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWC, in particular GEWF, notably F,    -   substitution of the isoleucine I in position 122 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular LAV, notably L,    -   substitution of the serine S in position 130 by a non-bulky        amino acid selected from the group consisting of GPLIVADCTN, in        particular PT, notably P,    -   substitution of the aspartic acid D in position 141 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE, in        particular SET, notably T,    -   substitution of the lysine K in position 164 by a polar amino        acid selected from the group consisting of WYSTCQNRHDE, in        particular NQR, notably N,    -   substitution of the glycine G in position 193 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCNQRKHDE, in particular ST, notably S,    -   substitution of the arginine R in position 223 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNKHDE, in particular CSTAH, notably AC,    -   substitution of the leucine L in position 226 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by apolar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular AIVH, notably V,    -   substitution of the leucine L in position 228 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VIMFGAPWYC, in particular IM, notably M,    -   substitution of the phenylalanine F in position 229 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN, in particular LTAS, notably S,    -   substitution of the leucine L in position 230 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN, in        particular PVA, notably P,    -   substitution of the cysteine C in position 258 by a hydrophobic        amino acid selected from the group consisting of VILMFGAPWY, in        particular YLIA, notably LA,    -   substitution of the isoleucine I in position 261 by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWYC, in particular FWC, notably F,    -   substitution of the tryptophane W in position 263 by a non-bulky        amino acid selected from the group consisting of GPLIVDCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPYC, in particular ALMFCITV, notably F,    -   substitution of the leucine L in position 274 by a hydrophobic        amino acid selected from the group consisting of VIMFGAPWYC, in        particular AVP, notably P,    -   substitution of the alanine A in position 275 by a hydrophobic        amino acid selected from the group consisting of VILMFGPWYC or        by a non-bulky amino acid selected from the group consisting of        GPLIVDCSTN, in particular NVMT, notably T,    -   substitution of the methionine M in position 280 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VLMFGAPWYC, in particular TYP, notably T,    -   substitution of the leucine L in position 282 by a bulky amino        acid selected from the group consisting of EKHRQYWFM, in        particular FMH, notably M,    -   and substitution of the serine S in position 297 by a polar        amino acid selected from the group consisting of WYTCQNRKHDE, in        particular QT, further comprises at least one mutation selected        from the group consisting of:    -   substitution of the isoleucine I in position 167 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VLMFGAPWYC, in particular VAL, notably V,    -   substitution of the aspartic acid D in position 191 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN or by a polar amino acid selected from the group        consisting of WYSTCQNRKHE, in particular ST, notably S,    -   substitution of the tyrosine Y position 257 by a non-bulky amino        acid selected from the group consisting of GPLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWC, in particular CSVW, notably C,    -   substitution of the cysteine C in position 259 a non-bulky amino        acid selected from the group consisting of GPLIVADSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWY, in particular SFWV, notably S,    -   substitution of the threonine T in position 260 a non-bulky        amino acid selected from the group consisting of GPLIVADCSN or        by a polar amino acid selected from the group consisting of        WYSCQNRKHE, in particular GH, notably G,    -   substitution of the aspartic acid D in position 262 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE or        by a non-bulky amino acid selected from the group consisting of        GPLIVADCSTN, in particular SLH,    -   substitution of the glycine G in position 264 non-bulky amino        acid selected from the group consisting of PLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFAPWYC, in particular AVP,    -   substitution of the threonine T in position 265 by a hydrophobic        amino acid selected from the group consisting of VILMFGAPWYC or        by a non-bulky amino acid selected from the group consisting of        GPLIVADCSN, in particular VWP, notably V,    -   substitution of the alanine A in position 266 by a polar amino        acid selected from the group consisting of WYSTCQNRKHDE, in        particular NQ, notably N,    -   substitution of the arginine R in position 267 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWYC, in particular IAP, notably IP,    -   substitution of the proline P in position 268 by a hydrophobic        amino acid selected from the group consisting of VILMFGAWYC, in        particular MCL, notably M,    -   substitution of the glutamic acid E in position 269 by a polar        amino acid selected from the group consisting of WYSTCQNRKHD, in        particular DQ, notably D,    -   substitution of the leucine L in position 270 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWC, in particular VA,    -   substitution of the lysine K in position 271 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular MLA,    -   substitution of the proline P in position 272 by non-bulky amino        acid selected from the group consisting of GPLIVADCSTN or by a        polar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular DEL, notably DL,    -   substitution of the lysine K in position 273 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRHD, in particular RP,    -   substitution of the proline P in position 276 by a bulky amino        acid selected from the group consisting of EHKRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAWYC, in particular KAV, notably K,    -   substitution of the arginine R in position 277 by a polar amino        acid selected from the group consisting of WYSTCQNKHDE, in        particular DNQ, notably DN,    -   substitution of the serine S in position 279 by a non-bulky        amino acid selected from the group consisting of GPLIVADCTN or        by a polar amino acid selected from the group consisting of        WYTCQNRKHDE, in particular GH,    -   substitution of the alanine A in position 281 by a non-bulky        amino acid selected from the group consisting of GPLIVDCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWYC, in particular ALV, notably AL,    -   and substitution of the isoleucine I in position 283 non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular VAL, notably V, further comprises at least one        mutation selected from the group consisting of:    -   substitution of the proline P in position 67 by a non-bulky        amino acid selected from the group consisting of GLIVADCSTN, in        particular GAV,    -   substitution of the glycine G in position 225 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN, in        particular AVP,    -   and substitution of the tryptophan W in position 278 by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPYC or by a charged amino acid selected from the group        consisting of RKHDEC, in particular RKDI, notably R.

It means that at least one substitution among the 29 particularsubstitutions of set 10 in position G7, K8, V27, F46, K54, T68, L72,176, V83, Y97, Y99, I122, S130, D141, K164, G193, R223, L226, L228,F229, L230, C258, 1261, W263, L274, A275, M280, L282 and N297 can beassociated with at least one substitution among the 21 particularsubstitutions of set 11 in position 1167, D191, Y257, C259, T260, D262,G264, T265, A266, R267, P268, E269, L270, K271, P272, K273, P276, R277,S279, A281 and 1283 and with at least one substitution among the 3particular substitutions of set 12 in position P67, G225 and W278.

The invention also relates to the above-mentioned mutatedhyperthermophilic PTE having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus islandicus corresponding to thesequence SEQ ID NO: 7, wherein the at least one mutation selected fromthe group consisting of:

-   -   substitution of the isoleucine I in position 167 by a non-bulky        amino acid selected from the group consisting of GPLVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VLMFGAPWYC, in particular VAL, notably V,    -   substitution of the aspartic acid D in position 191 by a        non-bulky amino acid selected from the group consisting of        GPLIVADCSTN or by a polar amino acid selected from the group        consisting of WYSTCQNRKHE, in particular ST, notably S,    -   substitution of the tyrosine Y position 257 by a non-bulky amino        acid selected from the group consisting of GPLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VLMFGAPWC, in particular CSVW, notably C,    -   substitution of the cysteine C in position 259 a non-bulky amino        acid selected from the group consisting of GPLIVADSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWY, in particular SFWV, notably S,    -   substitution of the threonine T in position 260 a non-bulky        amino acid selected from the group consisting of GPLIVADCSN or        by a polar amino acid selected from the group consisting of        WYSCQNRKHE, in particular GH, notably G,    -   substitution of the aspartic acid D in position 262 by a polar        amino acid selected from the group consisting of WYSTCQNRKHE or        by a non-bulky amino acid selected from the group consisting of        GPLIVADCSTN, in particular SLH,    -   substitution of the glycine G in position 264 non-bulky amino        acid selected from the group consisting of PLIVADCSTN or by a        hydrophobic amino acid selected from the group consisting of        VILMFAPWYC, in particular AVP,    -   substitution of the threonine T in position 265 by a hydrophobic        amino acid selected from the group consisting of VILMFGAPWYC or        by a non-bulky amino acid selected from the group consisting of        GPLIVADCSN, in particular VWP, notably V,    -   substitution of the alanine A in position 266 by a polar amino        acid selected from the group consisting of WYSTCQNRKHDE, in        particular NQ, notably N,    -   substitution of the arginine R in position 267 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWYC, in particular IAP, notably IP,    -   substitution of the proline P in position 268 by a hydrophobic        amino acid selected from the group consisting of VILMFGAWYC, in        particular MCL, notably M,    -   substitution of the glutamic acid E in position 269 by a polar        amino acid selected from the group consisting of WYSTCQNRKHD, in        particular DQ, notably D,    -   substitution of the leucine L in position 270 by a non-bulky        amino acid selected from the group consisting of GPIVADCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWC, in particular VA,    -   substitution of the lysine K in position 271 by a bulky amino        acid selected from the group consisting of EHRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPWYC, in particular MLA,    -   substitution of the proline P in position 272 by non-bulky amino        acid selected from the group consisting of GPLIVADCSTN or by a        polar amino acid selected from the group consisting of        WYSTCQNRKHDE, in particular DEL, notably DL,    -   substitution of the lysine K in position 273 by a non-bulky        amino acid selected from the group consisting of GPLIVADCSTN or        by a polar amino acid selected from the group consisting of        WYSTCQNRHD, in particular RP,    -   substitution of the proline P in position 276 by a bulky amino        acid selected from the group consisting of EHKRQYWFM or by a        hydrophobic amino acid selected from the group consisting of        VILMFGAWYC, in particular KAV, notably K,    -   substitution of the arginine R in position 277 by a polar amino        acid selected from the group consisting of WYSTCQNKHDE, in        particular DNQ, notably DN,    -   substitution of the serine S in position 279 by a non-bulky        amino acid selected from the group consisting of GPLIVADCTN or        by a polar amino acid selected from the group consisting of        WYTCQNRKHDE, in particular GH,    -   substitution of the alanine A in position 281 by a non-bulky        amino acid selected from the group consisting of GPLIVDCSTN or        by a hydrophobic amino acid selected from the group consisting        of VILMFGAPWYC, in particular ALV, notably AL,    -   and substitution of the isoleucine I in position 283 non-bulky        amino acid selected from the group consisting of GPLVADCSTN, in        particular VAL, notably V, further comprises at least one        mutation selected from the group consisting of:    -   substitution of the proline P in position 67 by a non-bulky        amino acid selected from the group consisting of GLIVADCSTN, in        particular GAV,    -   substitution of the glycine G in position 225 by a non-bulky        amino acid selected from the group consisting of PLIVADCSTN, in        particular AVP,    -   and substitution of the tryptophan W in position 278 by a        hydrophobic amino acid selected from the group consisting of        VILMFGAPYC or by a charged amino acid selected from the group        consisting of RKHDEC, in particular RKDI, notably R.

It means that at least one substitution among the 21 particularsubstitutions of set 11 in position I167, D191, Y257, C259, T260, D262,G264, T265, A266, R267, P268, E269, L270, K271, P272, K273, P276, R277,S279, A281 and 1283 can be associated with at least one substitutionamong the 3 particular substitutions of set 12 in position P67, G225 andW278.

A more particular subject of the invention is mutated hyperthermophilicPTE having a lactonase activity according to the present invention,derived from the hyperthermophilic PTE of Sulfolobus islandicuscorresponding to the sequence SEQ ID NO: 7, said mutatedhyperthermophilic PTE correspond to the following sequences:

-   -   SEQ ID NO: 95 corresponding to the SEQ ID NO: 7 comprising the        following one mutation: substitution of the tryptophan W in        position 263 by a phenylalanine F,    -   SEQ ID NO: 97 corresponding to the SEQ ID NO: 7 comprising the        following one mutation: substitution of the tryptophan W in        position 263 by a methionine M,    -   SEQ ID NO: 99 corresponding to the SEQ ID NO: 7 comprising the        following one mutation: substitution of the tryptophan W in        position 263 by a leucine L,    -   SEQ ID NO: 101 corresponding to the SEQ ID NO: 7 comprising the        following one mutation: substitution of the tryptophan W in        position 263 by an alanine A,    -   SEQ ID NO: 103 corresponding to the SEQ ID NO: 7 comprising the        following one mutation: substitution of the tryptophan W in        position 263 by an isoleucine I,    -   SEQ ID NO: 105 corresponding to the SEQ ID NO: 7 comprising the        following one mutation: substitution of the tryptophan W in        position 263 by a valine V,    -   SEQ ID NO: 107 corresponding to the SEQ ID NO: 7 comprising the        following one mutation: substitution of the tryptophan W in        position 263 by a threonine T,    -   SEQ ID NO: 109 corresponding to the SEQ ID NO: 7 comprising the        following three mutations: substitution of the cysteine C in        position 258 by a leucine L, substitution of the isoleucine I in        position 261 by a phenylalanine F, substitution of the        tryptophan W in position 263 by an alanine A,    -   SEQ ID NO: 111 corresponding to the SEQ ID NO: 7 comprising the        following four mutations: substitution of the valine V in        position 27 by an alanine A, substitution of the tyrosine Y in        position 97 by a tryptophan W, substitution of the leucine L in        position 228 by a methionine M, substitution of the tryptophan W        in position 263 by a methionine M,    -   SEQ ID NO: 113 corresponding to the SEQ ID NO: 7 comprising the        following four mutations: substitution of the valine V in        position 27 by an alanine A, substitution of the tyrosine Y in        position 97 by a tryptophan W, substitution of the tryptophan W        in position 263 by a leucine L, substitution of the methionine M        in position 280 by a threonine T,    -   SEQ ID NO: 115 corresponding to the SEQ ID NO: 7 comprising the        following four mutations: substitution of the cytosine C in        position 258 by an alanine A, substitution of the tryptophan W        in position 263 by a methionine M, substitution of the        methionine M in position 280 by a threonine T,    -   SEQ ID NO: 117 corresponding to the SEQ ID NO: 7 comprising the        following six mutations: substitution of the valine V in        position 27 by an alanine A, substitution of the isoleucine I in        position 76 by a threonine T, substitution of the tyrosine Y in        position 97 by a tryptophan W, substitution of the tyrosine Y in        position 99 by a phenylalanine F, substitution of the serine S        in position 130 by a proline P, substitution of the leucine L in        position 226 by a valine V,    -   SEQ ID NO: 119 corresponding to the SEQ ID NO: 7 comprising the        following six mutations: substitution of the leucine L in        position 72 by an isoleucine I, substitution of the tyrosine Y        in position 99 by a phenylalanine F, substitution of the        isoleucine I in position 122 by a leucine L, substitution of the        leucine L in position 228 by a methionine M, substitution of the        phenylalanine F in position 229 by a serine S, substitution of        the tryptophan W in position 263 by a leucine L,    -   SEQ ID NO: 121 corresponding to the SEQ ID NO: 7 comprising the        following seven mutations: substitution of the threonine T in        position 68 by a serine S, substitution of the leucine L in        position 72 by an isoleucine I, substitution of the serine S in        position 130 by a proline P, substitution of the leucine L in        position 228 by a methionine M, substitution of the        phenylalanine F in position 229 by a serine S, substitution of        the tryptophan W in position 263 by a methionine M, substitution        of the leucine L in position 274 by a proline P,    -   SEQ ID NO: 123 corresponding to the SEQ ID NO: 7 comprising the        following six mutations: substitution of the threonine T in        position 68 by a serine S, substitution of the isoleucine I in        position 76 by a threonine T, substitution of the tyrosine Y in        position 97 by a tryptophan W, substitution of the tyrosine Y in        position 99 by a phenylalanine F, substitution of the leucine L        in position 228 by a methionine M, substitution of the        tryptophan W in position 263 by a leucine L,    -   SEQ ID NO: 125 corresponding to the SEQ ID NO: 7 comprising the        following five mutations: substitution of the lysine K in        position 8 by an glutamic acid E, substitution of the tyrosine Y        in position 97 by a tryptophan W, substitution of the tyrosine Y        in position 99 by a phenylalanine F, substitution of the leucine        L in position 228 by a methionine M,    -   SEQ ID NO: 127 corresponding to the SEQ ID NO: 7 comprising the        following two mutations: substitution of the leucine L in        position 72 by an isoleucine I, substitution of the tryptophan W        in position 263 by a phenylalanine F,    -   SEQ ID NO: 129 corresponding to the SEQ ID NO: 7 comprising the        following five mutations: substitution of the threonine T in        position 68 by a serine S, substitution of the tyrosine Y in        position 97 by a tryptophan W, substitution of the tyrosine Y in        position 99 by a phenylalanine F, substitution of the serine S        in position 130 by a proline P, substitution of the leucine L in        position 228 by a methionine M,    -   SEQ ID NO: 131 corresponding to the SEQ ID NO: 7 comprising the        following four mutations: substitution of the valine V in        position 27 by an alanine A, substitution of the leucine L in        position 226 by a valine V, substitution the tryptophan W in        position 263 by a leucine L,    -   SEQ ID NO: 133 corresponding to the SEQ ID NO: 7 comprising the        following eight mutations: substitution of the proline P in        position 67 by a valine V, substitution of the threonine T in        position 68 by a serine S, substitution of the tyrosine Y in        position 97 by a tryptophan W, substitution of the tyrosine Y in        position 99 by a phenylalanine F, substitution of the leucine L        in position 228 by a methionine M, substitution of the cysteine        C in position 258 by an alanine A, substitution the tryptophan W        in position 263 by a leucine L, substitution of the methionine M        in position 280 by a threonine T,    -   SEQ ID NO: 135 corresponding to the SEQ ID NO: 7 comprising the        following eight mutations: substitution of the threonine T in        position 68 by a serine S, substitution of the tyrosine Y in        position 97 by a tryptophan W, substitution of the tyrosine Y in        position 99 by a phenylalanine F, substitution of the serine S        in position 130 by a proline P, substitution of the lysine K in        position 164 by an asparagine N, substitution of the leucine L        in position 226 by a valine V, substitution the tryptophan W in        position 263 by a methionine M,    -   SEQ ID NO: 137 corresponding to the SEQ ID NO: 7 comprising the        following five mutations: substitution of the threonine T in        position 68 by a serine S, substitution of the leucine L in        position 72 by an isoleucine I, substitution of the tyrosine Y        in position 97 by a tryptophan W, substitution of the tyrosine Y        in position 99 by a phenylalanine F, substitution of the serine        S in position 130 by a proline P.

The coding sequence of the above-mentioned mutated hyperthermophilic PTEhaving a lactonase activity according to the present invention, derivedfrom the hyperthermophilic PTE of Sulfolobus islandicus corresponding tothe sequence SEQ ID NO: 7 and corresponding to the following sequencesSEQ ID NO: 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, 122, 124, 126, 128, 130, 132, 134 and 136 are also part of theinvention.

The invention also related to mutated hyperthermophilic PTE having alactonase activity according to the present invention, derived from thehyperthermophilic PTE of Sulfolobus islandicus corresponding to thesequence SEQ ID NO: 7, said mutated hyperthermophilic PTE correspondingto the following sequences SEQ ID NO: 225, 227, 229, 231, 233, 235, 237,239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265 and267 for the proteins and to their respective coding sequences SEQ ID NO:224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250,252, 254, 256, 258, 260, 262, 264 and 266.

The invention relates more particularly to the above-mentioned mutatedhyperthermophilic PTE having a lactonase activity, derived from thehyperthermophilic PTE of Sulfolobus islandicus corresponding to thesequence SEQ ID NO: 7, further comprising at least one mutationcorresponding to a substitution of at least one of the amino acids ofthe following amino acid pairs, the positions of which in SEQ ID NO: 7are indicated hereafter, by another natural or non-natural amino acid:2R/314S, 14E/12E, 26R/75D, 26R/42E, 33R/42E, 33R/45E, 55R/52E, 55R/285E,74R/121D, 81K/42E, 81K/43D, 84K/80E, 109R/113E, 123K/162E, 147K/148D,151K/148D, 154R/150E, 154R/187E, 154R/188E, 161K/188E, 183R/150E,183R/187E, 183R/180E, 210K/245D, 215K/214D, 223R/256D, 223R/202D,234K/204D, 235R/202D, 241K/245D, 245D/244K, 250R/249D, 277R/286D,292K/298E, 310K/307E.

The invention also relates to a mutated hyperthermophilicphosphotriesterase having a lactonase activity derived from ahyperthermophilic phosphotriesterase defined by the consensus sequenceSEQ ID NO: 1, said mutated hyperthermophilic phosphotriesterase having asingle mutation being a substitution of the tryptophan W in position 265of the consensus sequence SEQ ID NO: 1.

In an embodiment, the invention relates to a mutated hyperthermophilicphosphotriesterase as defined above, said mutated hyperthermophilicphosphotriesterase having a single mutation being a substitution of thetryptophan W in position 265 of the consensus sequence SEQ ID NO: 1 by athreonine T.

In an embodiment, the invention relates to a mutated hyperthermophilicphosphotriesterase as defined above, said mutated hyperthermophilicphosphotriesterase having a single mutation being a substitution of thetryptophan W in position 263 of the sequence SEQ ID NO: 3 by anisoleucine I, a valine V, a threonine T or an alanine A.

The invention also relates to the isolated nucleic acid sequenceencoding the mutated hyperthermophilic PTE having a lactonase activityas defined above.

A subject of the invention is also the vectors comprising the nucleicacid encoding the mutated hyperthermophilic PTE having a lactonaseactivity as defined above. Such vectors can be plasmids, cosmids,phagemids or any other tool useful for cloning and expressing a nucleicacid.

The invention also relates to host cells, in particular bacteria,transformed by using the vector as defined above, such that their genomecontains nucleotide sequences encoding the mutated hyperthermophilic PTEhaving a lactonase activity as defined above, said mutatedhyperthermophilic PTE having a lactonase activity being produced in thecytoplasm of the host cells or secreted at their surface.

A subject of the invention is also is a method for generating a libraryof mutated hyperthermophilic PTE variants having a lactonase activitycomprising:

-   -   introducing into a population of host cells of a plurality of        vectors comprising a nucleic acid sequence encoding the mutated        hyperthermophilic PTE having a lactonase activity,    -   culturing the population of host cells in an appropriate culture        media,    -   expressing the polypeptide in the said cultured host cell,    -   recovering a plurality of mutated hyperthermophilic PTE        variants.

The invention also relates to a library of mutated hyperthermophilic PTEvariants having a lactonase activity obtainable by the method forgenerating a library of mutated hyperthermophilic PTE variants having alactonase activity as disclosed above.

The aim of said library is to provide polypeptide variants of mutatedhyperthermophilic PTE having a lactonase activity with enhancedphenotypic properties relative to those of the wild-typehyperthermophilic PTE having a lactonase activity from which theyderived.

The invention also relates to the use of a mutation to increase alactonase catalytic activity of a hyperthermophilic phosphotriesterasewhich has a sequence corresponding to the consensus sequence of SEQ IDNO: 1, wherein the amino acid W in position 265 is substituted by anamino acid chosen in the group consisting of the amino acids isoleucineI, valine V, threonine T or alanine A, to obtain a mutatedhyperthermophilic phosphotriesterase which has an increased lactonasecatalytic activity in comparison of the lactonase activity of saidhyperthermophilic phosphotriesterase corresponding to the consensussequence of SEQ ID NO: 1.

The invention also relates to the use of a single mutation to increase alactonase catalytic activity of a hyperthermophilic phosphotriesterasewhich has a sequence corresponding to the consensus sequence of SEQ IDNO: 1, wherein the amino acid W in position 265 is substituted by anamino acid chosen in the group consisting of the amino acids isoleucineI, valine V, threonine T or alanine A, to obtain a mutatedhyperthermophilic phosphotriesterase which has an increased lactonasecatalytic activity in comparison of the lactonase activity of saidhyperthermophilic phosphotriesterase corresponding to the consensussequence of SEQ ID NO: 1.

The invention also relates to a process for increasing a lactonasecatalytic activity of a hyperthermophilic phosphotriesterase which has asequence corresponding to the consensus sequence of SEQ ID NO: 1,comprising a step of substitution of the amino acid W in position 265 byan amino acid chosen in the group consisting of the amino acidsisoleucine I, valine V, threonine T or alanine A, to obtain a mutatedhyperthermophilic phosphotriesterase which has an increased lactonasecatalytic activity in comparison of the lactonase activity of saidhyperthermophilic phosphotriesterase corresponding to the consensussequence of SEQ ID NO: 1.

The invention also relates to a process for increasing a lactonasecatalytic activity of a hyperthermophilic phosphotriesterase which has asequence corresponding to the consensus sequence of SEQ ID NO: 1,comprising a step of a single substitution of the amino acid W inposition 265 by an amino acid chosen in the group consisting of theamino acids isoleucine I, valine V, threonine T or alanine A, to obtaina mutated hyperthermophilic phosphotriesterase which has an increasedlactonase catalytic activity in comparison of the lactonase activity ofsaid hyperthermophilic phosphotriesterase corresponding to the consensussequence of SEQ ID NO: 1.

The expression “a lactonase catalytic activity” refers to the hydrolysisof lactones, in particular N-acylhomoserine lactones (AHLs), whichmediate bacterial communication for many Gram negative bacteria and someArchaeal organisms.

The expression “an increased lactonase catalytic activity” means that,for the hydrolysis of an AHL, the mutated hyperthermophilic PTE has ahigher value of the ratio K_(cat)/K_(M) in comparison of the value ofthe ratio K_(cat)/K_(M) of the non-mutated hyperthermophilic PTE ofwhich it derives.

Preferably, K_(cat)/K_(M) of the mutated hyperthermophilic PTE isincreased of at least two times, more preferably between 25 and 70times, in comparison of the non mutated hyperthermophilic PTE.

In an embodiment, the invention concerns the use, or the process, asdefined above, wherein said hyperthermophilic phosphotriesterase is awild-type hyperthermophilic phosphotriesterase.

In an embodiment, the invention concerns the use, or the process, asdefined above, wherein hydrolyzis of 3-oxo-C12 AHL by said mutatedhyperthermophilic phosphotriesterase is increased by at least 2 times,in particular from 25 to 70 times, in comparison of hydrolyzis of3-oxo-C12 AHL by said hyperthermophilic phosphotriesterase.

Preferably, K_(cat)/K_(M) of the mutated hyperthermophilic PTE isincreased of at least two times, more preferably between 25 and 70times, in comparison of the non mutated hyperthermophilic PTE.

In an embodiment, the invention concerns the use, or the process, asdefined above, wherein said mutated hyperthermophilic phosphotriesterasehas a thermostability, which is substantially similar to thethermostability of said hyperthermophilic phosphotriesterase.

The expression “thermostability” refers to the ability of the PTE toresist to high temperatures, in particular above 70° C., moreparticularly between 70° C. and 120° C. At these temperatures, the 3Dstructure of the PTE is maintained, and these enzymes are still activeand able to hydrolyze OPs or lactones.

Classically, mutations which modify the catalytic activities of the PTEsare associated with a loss of the thermostability in the mutated PTE incomparison of the non-mutated PTE. However, the mutated PTEs of theinvention have a thermostability which is substantially similar to thethermostability of said hyperthermophilic phosphotriesterase.

The thermostability of the PTE can be verified by determining themelting temperature.

The melting temperature of the mutated PTE of the invention is higherthan 80° C., preferably higher than 85° C., preferably higher than 90°C.

This melting temperature can be measured by circular dichroismspectroscopy.

In an embodiment, the invention concerns the use, or the process, asdefined above, wherein the amino acid in position 2 in SEQ ID NO: 1 ismissing.

In an embodiment, the invention concerns the use, or the process, asdefined above, wherein said hyperthermophilic phosphotriesterase ischosen in the group consisting of SEQ ID NO: 3 from Sulfolobussolfataricus, SEQ ID NO: 5 from Sulfolobus acidocalaricus, or from SEQID NO: 7 Sulfolobus islandicus, wherein said sequences SEQ ID NO: 3, SEQID NO: 5 and SEQ ID NO: 7 belong to the consensus SEQ ID NO: 1, theamino acid in position 2 in SEQ ID NO: 1 being missing from SEQ ID NO: 5and the amino acids in position 2 and 3 in SEQ ID NO: 1 being missingfrom SEQ ID NO: 3 and SEQ ID NO: 7.

In an embodiment, the invention concerns the use, or the process, asdefined above, wherein at least the amino acid W in position 265 issubstituted by an amino acid chosen in the group consisting of the aminoacids isoleucine I, valine V, threonine T or alanine A.

In an embodiment, the invention concerns the use, or the process, asdefined above, wherein said amino acid W in position 265 is substitutedby an amino acid Isoleucine I.

In an embodiment, the invention concerns the use, or the process, asdefined above, wherein said mutated hyperthermophilic phosphotriesterasefurther comprises at least one additional substitution, said at leastone additional substitution being selected from the group consisting ofsubstitutions in positions G9, K10, V29, F/L48, K56, P69, T70, L74, 178,V85, 1124, L/S/N132, D143, K/N166, 1169, D193, G195, G227, L228, L230,F231, L232, Y259, C261, T262, 1263, D264, G266, T/I267, A268, K/R269,P270, E271, Y/L272, K273, P274, K275, L276, A277, P278, R/K279, W280,S281, I/M282, T/A/S283, L284, 1285, N/S/T299 of SEQ ID NO: 1.

In an embodiment, the invention concerns the use, or the process, asdefined above, wherein said mutated hyperthermophilic phosphotriesterasefurther comprises at least one additional substitution, said at leastone additional substitution being selected from the group consisting ofsubstitutions in positions G9, K10, V29, F/L48, K56, P69, T70, L74, 178,V85, I124, L/S/N132, D143, K/N166, I169, D193, G195, G227, L228, L230,F231, L232, Y259, C261, T262, 1263, D264, G266, T/I267, A268, K/R269,P270, E271, Y/L272, K273, P274, K275, L276, A277, P278, W280, S281,I/M282, T/A/S283, L284, 1285, N/S/T299 of SEQ ID NO: 1.

In an embodiment, the invention concerns the use, or the process, asdefined above, wherein said mutated hyperthermophilic phosphotriesterasefurther comprises at least one supplementary substitution, said at leastone supplementary substitution being selected from the group consistingof substitutions in positions Y99, Y101, R225 and C260 of SEQ ID NO: 1.

In an embodiment, the invention concerns the use, or the process, asdefined above, said mutated hyperthermophilic PTE corresponding to thefollowing sequences: SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ IDNO: 19, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107.

In an embodiment, the invention concerns the use, or the process, asdefined above, said mutated hyperthermophilic PTE corresponding to thefollowing sequences SEQ ID NO: 21, SEQ ID NO: 65 or SEQ ID NO: 109.

The invention also relates to compositions comprising the mutatedhyperthermophilic PTE having a lactonase activity as defined above.

In a preferred embodiment, the compositions as defined above comprisingthe mutated hyperthermophilic PTE having a lactonase activity furthercomprise at least one detergent.

In a more preferred embodiment, the above mentioned compositioncomprising both the mutated hyperthermophilic PTE having a lactonaseactivity and at least one detergent can be used as laundry detergent toclean up materials impregnated with OPs compounds.

An aspect of the invention concerns the use of the mutatedhyperthermophilic PTE of the invention for the decontamination of theorganophosphorous compounds. This aspect is based on the capacity of themutated hyperthermophilic PTE to catalyze the hydrolysis of phosphoesterbounds in OPs.

Therefore, the invention also relates to the use of a mutatedhyperthermophilic PTE having a lactonase activity as defined above, orof host cells as defined above, as bioscavengers:

-   -   within the context of the decontamination of the surfaces of        materials, of the skin or mucous membranes contaminated with        organophosphorous compounds, or    -   within the context of the prevention or treatment of an external        or of an internal poisoning by ingestion or inhalation of        organophosphorous compounds,    -   within the context of the pollution control of water polluted        with organophosphorus compounds, or    -   within the context of the destruction of stocks of neurotoxic        agents.

In an embodiment, the invention relates to the use as defined above,wherein said mutated hyperthermophilic PTE are chosen among the groupconsisting of: SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 23, SEQ ID NO:27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 53, SEQ ID NO: 55, SEQ IDNO: 67, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 97, SEQID NO: 99, SEQ ID NO: 111, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO:119.

In an embodiment, the invention relates to the use as defined above,wherein said mutated hyperthermophilic PTE are chosen among the groupconsisting of: SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 23, SEQ ID NO:27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 53, SEQ ID NO: 55, SEQ IDNO: 67, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 97, SEQID NO: 99, SEQ ID NO: 111, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO:119, SEQ ID NO: 21, SEQ ID NO: 65 and SEQ ID NO: 109.

A subject of the invention is also materials impregnated with mutatedhyperthermophilic PTE having a lactonase activity as defined above, inliquid or solid form, such as gloves, various garments, wipes, sprayfoams.

Another subject of the invention is kits of decontamination of thesurfaces of the materials, of the skins or mucous membranes,contaminated with organophosphorus compounds, or for the pollutioncontrol of water polluted with organophosphorus compounds, said kitcomprising mutated hyperthermophilic PTE having a lactonase activity asdefined above, or materials impregnated with mutated hyperthermophilicPTE having a lactonase activity as defined above.

A subject of the invention is also bioscavengers of organophosphoruscompounds comprising mutated hyperthermophilic PTE having a lactonaseactivity as defined above.

The invention also related to cartridges for external decontaminationinside which mutated hyperthermophilic PTE having a lactonase activityas defined above are grafted. Said cartridges can be used fordecontaminating the blood of an individual poisoned with OPs compounds.

Another aspect of the invention concerns the use of the mutatedhyperthermophilic PTE of the invention as antibacterial agents. Thisaspect is based on the capacity of the mutated hyperthermophilic PTE ofthe invention to hydrolyze lactones and, thus, to disrupt the quorumsensing of micro-organisms using homoserin lactone substrates tocommunicate.

Therefore, the invention concerns the use of a mutated hyperthermophilicphosphotriesterase as defined above to disrupt quorum-sensing inbacteria.

In an embodiment, the invention concerns the use of a mutatedhyperthermophilic phosphotriesterase, which has a sequence correspondingto the consensus sequence of SEQ ID NO: 1, wherein the amino acid W inposition 265 is substituted by an amino acid chosen in the groupconsisting of the amino acids isoleucine I, valine V, threonine T oralanine A, which has an increased lactonase catalytic activity incomparison of the lactonase activity of said hyperthermophilicphosphotriesterase corresponding to the consensus sequence of SEQ ID NO:1.

In an embodiment, the invention concerns the use of a mutatedhyperthermophilic phosphotriesterase as defined above to disruptquorum-sensing in bacteria, said mutated hyperthermophilic PTE beingchosen in the group consisting of: SEQ ID NO: 13, SEQ ID NO: 15, SEQ IDNO: 17, SEQ ID NO: 19, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQID NO: 63, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO:107.

In an embodiment, the invention concerns the use of a mutatedhyperthermophilic phosphotriesterase as defined above to disruptquorum-sensing in bacteria, said mutated hyperthermophilic PTE beingchosen in the group consisting of: SEQ ID NO: 21, SEQ ID NO: 65 and SEQID NO: 109.

In an embodiment, the invention concerns the use of a mutatedhyperthermophilic phosphotriesterase as defined above to disruptquorum-sensing in bacteria, said mutated hyperthermophilic PTE beingchosen in the group consisting of: SEQ ID NO: 9, SEQ ID NO: 11, SEQ IDNO: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 53, SEQID NO: 55, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75,SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 111, SEQ ID NO: 115, SEQ ID NO:117, SEQ ID NO: 119, SEQ ID NO: 21, SEQ ID NO: 65 and SEQ ID NO: 109.

In an embodiment, the invention concerns the use of a mutatedhyperthermophilic PTE of the invention to limit the formation ofbiofilms, notably in boats or other sea equipments.

In particular, a mutated hyperthermophilic PTE can be added to paintingmedia in order to limit the formation of biofilms, notably in boats orother sea equipments.

In an embodiment, the invention concerns the use of a mutatedhyperthermophilic PTE of the invention to inhibit the fire blight inplants or to inhibit the rotting of vegetables.

Fire blight in plants is due to infections by bacteria of the genusErwinia, whereas rotting of vegetables is due to infections by bacteriaof the genus Serratia.

Colonization of plants and vegetables by Erwinia and Serratia bacteriaboth involve a quorum sensing based on lactone substrates. Such lactonesubstrates can be hydrolysed by PTE to prevent and/or to treat Erwiniaand Serratia infections.

A subject of the invention is also a phytosanitary compositioncomprising as active ingredient at least one mutated hyperthermophilicPTE as defined above.

A subject of the invention is also an antibacterial compositioncomprising as an active ingredient at least one mutatedhyperthermophilic PTE as defined above.

The invention is also related to pharmaceutical compositions comprisingas active ingredient at least one mutated hyperthermophilic PTE having alactonase activity as defined above in combination with apharmaceutically acceptable vehicle.

The invention also relates to pharmaceutical compositions for their usein the treatment of pathology due to the presence of bacteria, notablypneumonia or nosocomial diseases.

The invention also relates to pharmaceutical compositions for their usein the treatment of dental plaque.

The invention also relates to pharmaceutical compositions for their useas eye drops in the treatment of eye infections or eye surface healing.

In a preferred embodiment, pharmaceutical compositions as defined abovecomprising the mutated hyperthermophilic PTE having a lactonase activityfurther comprise at least one antibiotic selected from the groupconsisting of gentamycine, ciprofloxacin, ceftazidime, imipenem,tobramycine.

In a more preferred embodiment, pharmaceutical compositions as definedabove are presented in a form which can be administered by injectableroute, in particular in solution or packaged or pegylated, or by topicalroute, in particular in the form of an ointment, aerosol or wipes.

The invention also related to use of materials impregnated with orcomprising the mutated hyperthermophilic PTE having a lactonaseactivity, as antiseptics for the decontamination of the surfacebacterial infection.

The invention also relates to compositions or pharmaceutical compositioncomprising the mutated hyperthermophilic PTE having a lactonase activityfor its use in the treatment of bacterial infections caused by bacteriausing homoserin lactone substrates to communicate, in particular in theblood, wounds, burn, skin, biomaterial-body contact area.

A subject of the invention is also a method for disrupting the quorumsensing of micro-organisms using homoserin lactone substrates tocommunicate, said method consisting of administering to a patient inneed thereof a sufficient amount of composition or pharmaceuticalcomposition comprising the mutated hyperthermophilic PTE having alactonase activity as defined above.

Another subject of the invention is also a mutated hyperthermophilic PTEas defined above for its use as a medicament.

In an embodiment, the invention concerns a mutated hyperthermophilic PTEas defined above, for its use in the treatment of bacterial infections.

In an embodiment, the invention concerns a mutated hyperthermophilic PTEas defined above for its use in the treatment of pneumonia or nosocomialdiseases, caused by bacteria using homoserin lactone substrates tocommunicate, in particular in the blood, wounds, burn, skin,biomaterial-body contact area.

In an embodiment, the invention concerns a mutated hyperthermophilic PTEas defined above for its use in the treatment of dental plaque.

In an embodiment, the invention concerns a mutated hyperthermophilic PTEas defined above for its use in the treatment of eye infections or eyesurface healing.

The invention is further illustrated by the figures and examples of thephosphotriesterase of Sulfolobus solfataricus, and mutations made to thelatter within the context of the preparation of mutatedhyperthermophilic PTE having a lactonase activity as defined aboveaccording to the invention. These examples are not intended to belimitation of the invention.

EXAMPLES Example 1

In this Example, SsoPox Variants have been Experimentally Produced andCharacterized.

1—Experimental Procedure 1.1—Initial Material

SsoPox coding gene is optimized for Escherichia coli expression and wassynthetized by GeneArt (Life Technologies, France)[1]. The gene wassubsequently cloned into a custom version of pET32b (Novagen)(=pET32b-ATrx-SsoPox) NcoI and NotI as cloning sites. The SsoPoxsequence has been verified by sequencage (Sequencage plateforme, Timone,Marseille, France). Both plasmids have been used for evolutionprotocols.

1.2—Site Directed Mutagenesis

A saturation site of position W263 of SsoPox was ordered to serviceprovider (GeneArt, Invitrogen; Germany) from the initially used plasmidpET22b-SsoPox. Each variant were checked by sequencing and provided asEscherichia coli DH5α cell glycerol stocks. The 20 plasmids(pET22b-SsoPox-W263X) have been purified from E. coli DH5a cells andtransformed into BL21(DE₃)-pLysS strain by electroporation for activityscreening and into BL21(DE₃)-pGro7/EL (TaKaRa) for high amountproduction/purification (see concerning section below).

For others site directed mutagenesis or saturation site of selectedpositions, pfu Turbo polymerase (Agilent) has been used to amplify theoverall plasmid using primers incorporating wanted variations. PCRcomposition has been performed as advised by the customer in a finalvolume of 25 μL and amplification was performed from 100 ng of plasmid.The PCR protocol was the following:

95° C. 10′ 1X 95° C. 45″ 30X  50° C.  1′ 68° C. 15′ 68° C. 20′ 1X 14° C.∞ 1X

Remaining initial plasmids were removed by DpnI enzymatic digestion (11;Fermentas) during 45′ at 37° C. After inactivation of 20′ at 90° C., DNAwas purified (QIAquick PCR Purification Kit; Qiagen) to obtain about 30μL of variable amount of DNA. 5 μL of purified DNA was then transformedinto Escherichia coli electrocompetent cells (50 μL; E. cloni; Lucigen),recovered in 1 mL of SOC medium during 1h at 37° C. and then plated onagar medium supplemented with ampicillin (100 μg/mL). Several cloneswere sequenced to verify the well-performed mutagenesis (Sequencageplateforme, Timone, Marseille, France) and verified plasmids weretransformed into E. coli strain BL21(DE₃)-pGro7/GroEL (TaKaRa) for highamount production/purification and analysis (see concerning sectionbelow).

1.3—Directed Evolution Process

Directed evolution protocol has been performed using the GeneMorph® IIRandom Mutagenesis Kit in 25 aL final, using primers T7-promotor (TAATAC GAC TCA CTA TAG GG) and T7-RP (GCT AGT TAT TGC TCA GCG G) and 500 ngof matrix (correspond to 6 μg of pET32b-ΔTrx-SsoPox plasmid). Others PCRelements have been performed as advised by the customer recommendations.The PCR protocol was the following:

95° C.  5′ 1X 95° C. 30″ 30X  55° C. 30″ 72° C.  4′ 72° C. 10′ 1X 14° C.∞ 1X

Remaining plasmid was then digested by DpnI enzyme (1 μl; Fermentas)during 45′ at 37° C. and then inactivated 20′, 90° C. DNA was thenpurified (QIAquick PCR Purification Kit; Qiagen) to obtain about 50 μLof DNA at 100 ng/μL. For the next steps please refer to part “clonageand bank generation”.

1.4—Method

SsoPox coding gene has been amplified from pET32b-ATrx-SsoPox plasmid byPCR (500 μL RedTaq; Sigma) using primers T7-promotor (TAA TAC GAC TCACTA TAG GG) and T7-RP (GCT AGT TAT TGC TCA GCG G). The PCR protocol wasthe following:

95° C.  2′ 1X 95° C. 30″ 25X  55° C.  1.5′ 72° C.  1.2′ 72° C.  7′ 1X16° C. ∞ 1X

Remaining plasmid was then digested by DpnI enzyme (1 μl; Fermentas)during 45′ at 37° C. and then inactivated 20′, 90° C. DNA was thenpurified (QIAquick PCR Purification Kit; Qiagen) to obtain about 100 μLof DNA at 200 ng/μL. 15 μL of DNA (˜3 μg) was digested by 2 UE of DNAseI(TaKaRa) in buffer TrisHCl 100 mM pH 7.5, MnCl₂ 10 mM at 20° C. during30″, 1′ and 2′. Digestions were stopped by 10′ incubation at 90° C. inpresence of EDTA 60 mM. After spin down, DNA aliquots were pooled andrun on electrophoresis agarose (2%; w/v) gel in TAE buffer during 15′ at50 mA. Fragments consisting of average size of 70 bp (from 50 to 150 pb)were excised from gel and purified using D-tube™ Dyalizer Maxi(Calbiochem) devices.

DNA extracted from gel (concentration >12 ng/VL) was used as matrix in“assembly PCR” consisting of 100 ng of matrix, 2 pmol of primersincorporating mutations and using 2.5 UE of Pfu Turbo polymerase(Agilent) with a final volume of 25 al. The primer mix was composed ofan oligonucleotide mix consisting of equivalent amount of modifiedpositions. The PCR protocol was the following:

94° C. 2′   1X 94° C. 30″  35X  65° C. 1.5′ 62° C. 1.5′ 59° C. 1.5′ 56°C. 1.5′ 53° C. 1.5′ 50° C. 1.5′ 47° C. 1.5′ 45° C. 1.5′ 41° C. 1.5′ 72°C. 45″  72° C. 7′   1X  4° C. ∞ 1X

The primer incorporating mutations in the directions 5′-3′ are asfollows:

TABLE 1 Listing of primers used to create SsoPox variants SEQ ID NOPrimer Sequence 5′-3′ SEQ ID NO: 268 W263M-FTGCACCATTGATATGGGCACCGCAAAACCG SEQ ID NO: 269 W263M-RCGGTTTTGCGGTGCCCATATCAATGGTGCA SEQ ID NO: 270 W263L-FTGCACCATTGATCTGGGCACCGCAAAACCG SEQ ID NO: 271 W263L-RCGGTTTTGCGGTGCCCAGATCAATGGTGCA SEQ ID NO: 272 W263A-FTGCACCATTGATGCAGGCACCGCAAAACCG SEQ ID NO: 273 W263A-RCGGTTTTGCGGTGCCTGCATCAATGGTGCA SEQ ID NO: 274 W263I-FTGCACCATTGATATTGGCACCGCAAAACCG SEQ ID NO: 275 W263I-RCGGTTTTGCGGTGCCAATATCAATGGTGCA SEQ ID NO: 276 W263V-FTGCACCATTGATGTTGGCACCGCAAAACCG SEQ ID NO: 277 W263V-RCGGTTTTGCGGTGCCAACATCAATGGTGCA SEQ ID NO: 278 W263T-FTGCACCATTGATACCGGCACCGCAAAACCG SEQ ID NO: 279 W263T-RCGGTTTTGCGGTGCCGGTATCAATGGTGCA SEQ ID NO: 280 C258L-FATTAGCCATGATTATCTGTGCACCATTGAT SEQ ID NO: 281 C258L-RATCAATGGTGCACAGATAATCATGGCTAAT SEQ ID NO: 282 I261F-FGATTATTGCTGCACCTTTGATTGGGGCACC SEQ ID NO: 283 I261F-RGGTGCCCCAATCAAAGGTGCAGCAATAATC SEQ ID NO: 284 V27A-FGAACATCTGCGTGCATTTAGCGAAGCAGTT SEQ ID NO: 285 V27A-RAACTGCTTCGCTAAATGCACGCAGATGTTC SEQ ID NO: 286 Y97W-FGGCACCGGTATTTGGATTTATATCGATCTGCCG SEQ ID NO: 287 Y97W-RCGGCAGATCGATATAAATCCAAATACCGGTGCC SEQ ID NO: 288 L228M-FGATCGTTATGGTCTGGACATGTTTCTGCCGGTT SEQ ID NO: 289 L228M-RAACCGGCAGAAACATGTCCAGACCATAACGATC SEQ ID NO: 290 I280T-FGCACCGCGTTGGAGCACTACCCTGATTTTTG SEQ ID NO: 291 I280T-RCAAAAATCAGGGTAGTGCTCCAACGCGGTGC SEQ ID NO: 292 F46L-FCTGTATAATGAAGATGAAGAACTGCGCAATGCCGTGAATGAAG SEQ ID NO: 293 F46L-RCTTCATTCACGGCATTGCGCAGTTCTTCATCTTCATTATACAG SEQ ID NO: 294 I76T-FGTTATGGGTCTGGGTCGTGATACTCGTTTTATGGAAAAAGTTGTG SEQ ID NO: 295 I76T-RCACAACTTTTTCCATAAAACGAGTATCACGACCCAGACCCATAAC SEQ ID NO: 296 Y99F-FGGCACCGGTATTTATATTTTTATCGATCTGCCG SEQ ID NO: 297 Y99F-RCGGCAGATCGATAAAAATATAAATACCGGTGCC SEQ ID NO: 298 L130P-FGGCATTCAGGGCACCCCGAATAAAGCAGGTTTTG SEQ ID NO: 299 L130P-RCAAAACCTGCTTTATTCGGGGTGCCCTGAATGCC SEQ ID NO: 300 L226V-FGATCGTTATGGTGTGGACCTGTTTCTGCCGGTT SEQ ID NO: 301 L226V-RAACCGGCAGAAACAGGTCCACACCATAACGATC SEQ ID NO: 302 L72I-FGTTATGGGTATTGGTCGTGATATTCGTTTT SEQ ID NO: 303 L72I-RAAAACGAATATCACGACCAATACCCATAAC SEQ ID NO: 304 F229S-FGATCGTTATGGTCTGGACCTGTCTCTGCCGGTT SEQ ID NO: 305 F229S-RAACCGGCAGAGACAGGTCCAGACCATAACGATC SEQ ID NO: 306 T68S-FAAAACCATTGTTGATCCGAGTGTTATGGGT SEQ ID NO: 307 T68S-RACCCATAACACTCGGATCAACAATGGTTTT SEQ ID NO: 308 K8E-FCATTCCGCTGGTTGGTGAAGATAGCATTGAAAG SEQ ID NO: 309 K8E-RCTTTCAATGCTATCTTCACCAACCAGCGGAATG SEQ ID NO: 310 P67S-FAAAACCATTGTTGATTCGACCGTTATGGGT SEQ ID NO: 311 P67S-RACCCATAACGGTCGAATCAACAATGGTTTT SEQ ID NO: 312 K164N-FCAATAAAGAAACCAATGTTCCGATTATTACCC SEQ ID NO: 313 K164N-RGGGTAATAATCGGAACATTGGTTTCTTTATTG

Finally, assembly PCR was used as matrix for “nested PCR”. 1 μL ofassembly PCR was used as classical PCR (50 μL, RedTaq; Sigma) withcloning primers SsoPox-lib-pET-5′ (ATGCGCATTCCGCTGGTTGG) andSsoPox-lib-pET-3′ (TTATTAGCTAAAGAATTTTTTCGGATTTTC). The PCR protocol wasthe following:

95° C.  2′ 1X 95° C. 30″ 25X  65° C.  1.5′ 1X 72° C.  7′ 16° C. ∞ 1X

1.5—Clonage and Bank Generation

PCR product has been purified using extraction kit (QIAquick PCRPurification Kit; Qiagen) and then digested for 45′ at 37° C. by NcoIFastdigest and NotI Fastdigest enzymes (12UE of each enzyme; Fermentas).Enzymes were then inactivated by 20′ incubation at 90° C. and thenpurified (QIAquick PCR Purification Kit; Qiagen) to be cloned intopET32b-Δtrx plasmid at the corresponding restriction sites previouslydephosphorylated as recommended by the customer (10 UE/μl CIP; NEB).Ligation has been performed in a molar ratio 1:3 with 50 ng of plasmidusing T4-DNA ligase during 16h at 16° C. (20 UE; NEB).

After ligation, ligase was inactivated 20′ at 90° C. and then purifiedfrom salts by classical alcohol precipitation and recovered in 10 μL ofwater. Escherichia coli electrocompetent cells (50 μL; E. cloni;Lucingen) were electroporated with 5 μL of purified ligation andrecovered in 1 mL of SOC medium for 1h at 37° C. All 1 mL was thenplated on agar selected medium (ampicillin 100 μg/mL) and incubatedovernight at 37° C.

Obtaining transformation efficiency higher than 10⁴ colonies on agarplate, the colonies were then harvested using 1 mL of plasmidicextraction kit solution 1 (Qiaprep Spin Miniprep kit; Quiagen) andplasmids were then extracted from cells following the recommendedprocedure. The plasmid pool obtained constituting the bank, 100 ng wereused to electroporate 50 μL of electrocompetent BL21(DE3)-pGro7/EL(TaKaRa). After 1h of recovering in SOC medium at 37° C., cells wereplated on agar plate added of ampicillin (100 ag/mL) and chloramphenicol(37 ag/mL).

2—Screening Procedure

Microcultures consisting of 600 μL of ZYP medium [3,4] supplemented byampicillin (100 μg/mL) and chloramphenicol (34 ag/mL) are inoculated bya tip picked colony in 96 well plates. Cultures grew at 37° C. under 1600 rpm agitation for 5h before activation mediated by temperaturetransition to 25° C. and addition of CoCl₂ (0.2 mM) and arabinose (0.2%,w/v). After overnight growth, tips were removed and used to pickseparated colony on agar plate (ampicilin 100 μg/mL; chloramphenicol 34μg/mL) for strain conservation. Cultures were centrifuged to keep cellpellets which were resuspended in lysis buffer consisting of 50 mM HEPESpH 8, 150 mM NaCl, CoCl₂ 0.2 mM, Lysozyme 0.25 mg/ml, PMSF 0.1 mM DNAseI10 ag/ml and MgSO₄ 20 mM. Cells were disrupted by freezing/thawing stepsand cells debris were removed by centrifugation (13 000 g, 4° C., 30′).Partial purification of the protein was performed exploiting SsoPoxhyperthermostability [5] by 15 minutes incubation at 70° C. Aggregatedproteins were harvested by centrifugation (13 000 g, 25° C., 30′).

2.1—Phosphotriesterase Activity Screening

Phosphotriesterase activity screening was mediate by monitoringchromophoric phosphotriester hydrolysis (paraoxon, methyl-paroxon,parathion, methyl parathion (1 mM or 100 μM, Fluka). Kineticsexperiments were performed for 10′monitoring phosphotriester(ε_(405 nm)=17 000 M⁻¹ cm⁻¹) hydrolysis at 25° C. using a microplatereader (Synergy HT; BioTek, USA) and the Gen5.1 software in a 6.2 mmpath length cell for 200 μL reaction in 96-well plate. Standard assayswere performed in pte buffer (50 mM HEPES pH 8, 150 mM NaCl, 0.2 mMCoCl₂).

2.2—Lactonase Activity Screening

Lactonase activity screening was mediated by a genetically modifiedstrain PAO1 of Pseudomonas aeruginosa (PAO1-ΔlasI-JP2). The JP2 plasmidencodes proteins coding for bioluminescence production in presence of3-oxo-C12 AHLs in P. aeruginosa; the lasI gene, responsible of 3-oxo-C12AHLs synthesis in wt P. aeruginosa, is deleted. SsoPox variants (5 μL oftenfold diluted partially purified variants) are mixed in 100 μL of ptebuffer with 3-oxo-C12 AHL (100 nM) and incubated 20 minutes at roomtemperature. A volume of 450 μL of LB media (Trimethoprime lactate 300ag/mL) was inoculated by overnight preculture of P. aeruginosaPAO1-ΔlasI-JP2 (1/50) and supplemented with the mixture protein/AHLs (50μL). The final theoretical concentration of 3-oxo-C12 AHLs is 20 nM,prior to enzymatic hydrolysis by SsoPox. After 270 minutes of culture at37° C., cell density (OD_(600 nm)) and bioluminescence (460-40 nm;intensity 100) of 200 μL aliquots of culture are measured in a 96-wellplate using a microplate reader (Synergy HT, BioTek, USA) monitored byGen5.1 software. Controls consist in the same experiment without enzymeand/or without AHLs.

Best hits were re-plated and then placed in microcultures as previouslyexplained despite each clones were represented four times. The previousprotocol was performed as identic to confirm the results. However, lysisbuffer and pte buffer doesn't contain CoC1₂ salt to avoid affinity lossfor the metals by the enzyme during the improvement process.

3—Improvement Confirmation and Analysis

The best variants were then sequenced (Sequencage plateforme, Timone,Marseille, France) and produce in larger amount for catalytic propertiesanalysis. Genes or plasmids selected for the best improvement can havebeen used to perform the next round of diversity generation (i.e. goback to the first sections).

The high amount of protein production was performed using E. coli strainBL21(DE₃)-pGro7/GroEL (TaKaRa). Productions have been performed in 500mL of ZYP medium [3] (100 ag/ml ampicilline, 34 ag/ml chloramphenicol)as previously explained [4,6,7], 0.2% (w/v) arabinose (Sigma-Aldrich;France) was added to induce the expression of the chaperones GroEL/ESand temperature transition to 25° C. was perfomed. Purification wasperformed as previously explained [7]. Briefly, a single step of 30′incubation at 70° C. was performed, followed by differential ammoniumsulfate precipitation, dialysis and exclusion size chromatography.Proteins were quantified using nanospectrophotometer (nanodrop,thermofisher scientific, France) using protein molar extinctioncoefficient generated using protein primary sequence in PROT-PARAM(expasy tool softwares)[8].

3.1—Kinetics Generalities

Catalytic parameters were evaluated at 25° C., and recorded with amicroplate reader (Synergy HT, BioTek, USA) and the Gen5.1 software in a6.2 mm path length cell for 200 μL reaction in 96-well plate aspreviously explained [6]. Catalytic parameters were obtained by fittingthe data to the Michaelis-Menten (MM) equation [9] using Graph-Pad Prism5 software. When V_(max) could not be reached in the experiments, thecatalytic efficiency was obtained by fitting the linear part of MM plotto a linear regression using Graph-Pad Prism 5 software.

3.2—Phosphotriesterase Activity Characterization

Standard assays were performed in pte buffer measuring time coursehydrolysis of PNP derivative of OPs (ε_(405 nm)=17 000 M⁻¹ cm⁻¹), nerveagents coumarin derivatives (CMP-coumarin, IMP-coumarin,PinP-coumarin)[10](ε_(412 nm)=37 000 M⁻¹cm⁻¹) or malathion bu adding 2mM DTNB in the buffer (ε_(412 nm)=13 700 M⁻¹ cm⁻¹). Kinetics have alsobeen performed in pte buffer added of 0.1 and/or 0.01% of SDS aspreviously exemplified [1].

3.3—Lactonase Activity Characterization

The lactonase kinetics were performed using a previously describedprotocol [6]. The time course hydrolysis of lactones were performed inlac buffer (Bicine 2.5 mM pH 8.3, NaCl 150 mM, CoCl₂ 0.2 mM, Cresolpurple 0.25 mM and 0.5% DMSO) over a concentration range 0-2 mM forAHLs. Cresol purple (pK_(a) 8.3 at 25° C.) is a pH indicator used tofollow lactone ring hydrolysis by acidification of the medium. Molarcoefficient extinction at 577 nm was evaluated recording absorbance ofthe buffer over an acetic acid range of concentration 0-0.35 mM.

3.4—Melting Temperature Determination

Circular Dichroism spectra were recorded as previously explained [6]using a Jasco J-810 spectropolarimeter equipped with a Pelletier typetemperature control system (Jasco PTC-4235) in a 1 mm thick quartz celland using the Spectra Manager software. Briefly, measurements wereperformed in 10 mM sodium phosphate buffer at pH 8 with a proteinconcentration of 0.1 mg/mL. Denaturation was recorded at 222 nm byincreasing the temperature from 20 to 95° C. (at 5° C./min) in 10 mMsodium phosphate buffer at pH 8 containing increasing concentrations(1.5-4 M) of guanidinium chloride. The theoretical Tm withoutguanidinium chloride was extrapolated by a linear fit using theGraphPadPrism 5 software.

2—Results

2.1—Phosphotriesterase and Lactonase Activity Screening

It has been previously highlighted that some residues in SsoPox activesite are deleterious for phosphotriesterase activity compared to P.diminuta PTE active site (Hiblot et al., 2012, PloS One 7(10), e47028).In particular, W263 make a steric hindrance in SsoPox active siteblocking the entry of OPs. However, it has been shown that W263Fvariation allowed a phosphotriesterase activity improvement despite thatTrp and Phe are both cumbersome residues⁵. This raises the question ofthe real impact of variation at position W263. Indeed, W263 position islocated at the dimer interface and on the active site capping looppositioning the lactone ring in SsoPox complexed structure with HTL⁶.Thus, variations at this position have been study to better understandtheir structural impacts allowing activity improvement.

Phosphotriesterase and Lactonase Activities Screening

A saturation site of the W263 position has been performed in the aim toscreen phosphotriesterase and lactonase activities. Each variant havebeen produced in small amount (3 mL) and partially purified exploitingthe natural thermoresistance of SsoPox to perform activity screening.

Phosphotriesterase Activity Screening

The ability of each variant to hydrolyse paraoxon substrate (FIGS.1(A)-1(F)) has been evaluated with 1 mM and 100 μM with same tendenciesobserved. The most efficient variants were respectively SsoPox W263L,W263M and W263F with specific activities enhancements ranging between30-50 and 20-35 times respectively at 1 mM and 100 μM, compared toSsoPox wt. Moreover, it is interesting to note that the native enzyme(W263) is the less efficient among the saturation site variants forparaoxon hydrolysis (after W263K).

CMP-coumarin (FIG. 1(C)) is a cyclosarin derivative used also toevaluate the ability of variants to hydrolyse nerve agents (50 μM). TheSsoPox variants W263F, W263M and W263L exhibit the best specificactivities (FIG. 2(C)). The improvements range between 4 and 11 timescompared to SsoPox wt.

In conclusion, SsoPox W263F, W263L and W263M seem the best able toimprove phosphotriesters hydrolysis. Variations implicate mainlyhydrophobic residues with variable cumbersome. These proteins will formthe group “phosphotriesterase selected variants”.

Lactonase Activity Screening

It has been postulated that reduction of steric hindrance is not theonly explanation for phosphotiresterase activity improvement of SsoPoxW263F. So, variation at this position could allow a lactonase activityimprovement. In the aim to explore this issue, a lactonase activityscreening method has been developed. This screen is based on P.aeruginosa PAO1 derivative strain deleted for lasI gene and carrying aJP2 plasmid allowing to produce bioluminescence in presence of 3-oxo-C12AHLs (1) (FIG. 1(C)) (a main lactone implicated in P. aeruginosa quorumsensing system). In few words, this strain doesn't produce by itself3-oxo-C12 AHLs and, thus, doesn't generate intrinsicallybioluminescence. The bioluminescence intensity in experiment is only dueto exogenously added 3-oxo-C12 AHLs. Thus, if a lactonase ispre-incubated with 3-oxo-C12 AHLs, the bioluminescence will be inverselyproportional to lactonase (3-oxo-C12 AHLase) activity.

Using this screening method, SsoPox W263A, W263I, W263T and W263V havebeen selected for a potential lactonase activity improvement (FIG.3(B)). The amplitude of the ameliorations in our conditions of screeningcan't be evaluated. Selected variations implicate small and mainlyhydrophobic residues. These four variants form the group “lactonaseselected variants”.

2.2—Enzymatic Characterization of SsoPox Variants

2.2.1—Single Position Variants

Confirmation of screening results has been allowed by enzymaticcharacterisation of phopshotriesterase and lactonase selected variants.They have been produced and purified in large amount. Their catalyticparameters have been characterized for lactone (3-oxo-C12 AHL (1)) andphosphotriester (Paraoxon) substrates (Table 2).

TABLE 2 Lactonase and phosphotriesterase activity of W263 variants ofSsoPox (ND corresponds to not determined value). SsoPox variant k_(cat)(s⁻¹) K_(M) (μM) K_(I) (μM) k_(cat)/K_(M) (M⁻¹s⁻¹) Enhancement/wtParaoxon Wt 12.59 ± 1.26 24250 ± 3716 — 5.19(±1.31) × 10² 1 W263F  8.47± 0.53  700 ± 146 — 1.21(±0.33) × 10⁴ 23.3 W263M  6.82 ± 0.57  931 ± 163—  7.33(±1.9) × 10³ 14.1 W263L ND ND — 2.37(±0.33) × 10³ 4.6 W263I ND ND— 1.21(±0.59) × 10³ 2.3 W263V ND ND —  8.83(±0.3) × 10² 1.7 W263T ND ND— 1.06(±0.03) × 10³ 2.0 W263A  5.29 ± 0.69  1491 ± 351 — 3.55(±1.30) ×10³ 6.8 3-oxo-C12 AHL (l) Wt (9.9 ± 1.2) × 10⁻¹   335 ± 10.4 —2.96(±0.99) × 10³ 1 W263F (6.6 ± 0.3) × 10⁻¹   146 ± 33 — 4.52(±1.04) ×10³ 1.5 W263M ND ND ND ND 0 W263L ND ND ND ND 0 W263I  2.89 ± 0.08  17.8± 4.87 — 1.62(±0.45) × 10⁵ 54.7 W263V  4.82 ± 0.11  24.7 ± 5.2 —1.95(±0.44) × 10⁵ 65.8 W263T  10.4 ± 0.35   137 ± 19 — 7.56(±1.08) × 10⁴25.6 W263A  20.4 ± 1.21   1640 ± 170 — 1.25(±0.15) × 10³ 0.42

Phosphotriesterase Activity Characterization

Phosphotriesterase activity of wt SsoPox has been characterized in aprevious study (Hiblot et al., 2012, PloS One 7(10), e47028). As alreadyobserved in screening experiments, catalytic efficiencies of allselected variants were higher than the wt protein for paraoxon (Table2). Among the phosphotriesterase selected variants, SsoPox W263Fexhibits the highest paraoxonase catalytic efficiency(k_(cat)/K_(M)=1.21(±0.33)×10⁴ M⁻¹s⁻¹) followed by SsoPox W263M andSsoPox W263L with respective enhancements of 23.3, 14.1 and 4.6 timescompared to wt enzyme (Table 2, FIG. 2(D)). Among the lactonase selectedvariants, only SsoPox W263A (k_(cat)/K_(M)=3.55(±1.30)×10³ M⁻¹s⁻¹)exhibits a higher paraoxonase catalytic efficiency than the SsoPox W263Lphosphotriesterase selected variant (6.8 times improvement compared towt enzyme).

SsoPox W263L variant was selected for its phosphotriesterase activityimprovement. Owing its potential for phosphotriester hydrolysis, itsability to hydrolyze different nerve agent derivatives has beenaddressed (Table 3).

TABLE 3 Phosphotriesterase activity of W263L variant of SsoPox. SsoPoxW263L k_(cat) (s⁻¹) K_(M) (μM) K_(I) (μM) k_(cat) /K_(M)(M⁻¹s⁻¹)Paraoxon 3.13 ± 0.25  985 ± 169 — 3.18 (±0.60) × 10³ Paraoxon + SDS0.01% 8.89 ± 0.99 141 ± 33 1700 ± 453 6.29 (±1.62) × 10⁴ Paraoxon + DOC0.01% 3.17 ± 0.18 244 ± 55 — 1.30 (±0.30) × 10⁴ Methyl-paraoxon ND ND ND3.16 (±0.10) × 10⁴ Methyl-paraoxon + SDS ND ND ND 1.69 (±0.04) × 10⁵0.01% Methyl-parathion + SDS 1.22 (±0.13) × 10⁻¹ 168 ± 31 1920 ± 676 728± 156 0.01% ND corresponds to not determined value. For paraoxon andmethyl-parathion in presence of SDS, experimental data were fitted tosubstrate inhibition equation because of a more suitable fit than withclassical MM equation. As a consequence, the calculated catalyticefficiencies are available only at low substrate concentration.

It has been shown that anionic detergents, like SDS, were able toenhance the phosphotriesterase activity of SsoPox (Hiblot et al., 2012,PloS One 7(10), e47028). Paraoxon hydrolysis by SsoPox W263L in presenceof SDS at 0.01% (k_(cat)/K_(M)=6.29(±1.62)×10⁴ M⁻¹s⁻¹) has been comparedto the paraoxon hydrolysis by wt enzyme (k_(cat)/K_(M)=6.41(±1.51)×10³M⁻¹s⁻¹). The catalytic efficiency improvement induced by SDS on SsoPoxW263L (19.8 times) is higher than one observed for wt SsoPox (12.4times). It was proposed that activity improvement by SDS is due toglobal flexibilisation of the protein (Hiblot et al., 2012, PloS One7(10), e47028). The higher improvement observed for SsoPox W263L couldbe due to variation-induced flexibility mimicking partially theSDS-induced flexibility. Indeed, leucine being less cumbersome than Trp,the phosphotriesterase improvement can be imputed to steric hindrancereduction.

Moreover, SDS at 0.01% is also able to enhance methyl-paraoxonhydrolysis by SsoPox W263L (5.3 times).

Deoxycholate acid (DOC), a mild detergent, is less effective than theSDS in increasing the paraoxon hydrolysis by the SsoPox W263L(k_(cat)/K_(M)=1.30(±0.30)×10⁴).

Lactonase Activity Characterization

Chemically different lactone substrates have been used in the aim tounderstand the lactonase activity improvement of SsoPox. AHLs andγ/δ-lactones (oxo-lactones) are differently acylated on the lactonecycle (FIGS. 1(A)-1(F)). We have studied AHLs with different sizechains. 3-oxo-C10 AHLs (l) (FIG. 1(C)) are 10 times better substrate forwt enzyme compared to 3-oxo-C12 AHL (1) (respectively,k_(cat)/K_(M)=3.16(±0.40)×10⁴ M⁻¹s⁻¹ and k_(cat)/K_(M)=2.96(±0.99)×10³M⁻¹s⁻¹; data not shown) for which variants have been screened. Twodifferent oxo-lactones exhibiting different ring sizes have also beenstudied. Wt SsoPox exhibits a 10 times higher catalytic efficiency withundecanoic-δ-lactones (r) (6 atoms ring-size) than withundecanoic-γ-lactones (r) (5 atoms ring-size) (respectively,k_(cat)/K_(M)=6.72(±2.54)×10⁴ M⁻¹s⁻¹ and k_(cat)/K_(M)=2.36 (±0.38)×10³M⁻¹s⁻¹; data not shown).

Directed evolution allows to “select what you screen for”. Giving thatbest lactonase variants were selected on their ability do hydrolyse3-oxo-C12 AHL, kinetic characterisations of the 3-oxo-C12 AHLaseactivity has been performed (Table 2). Results obtained allows toconfirm that lactonase selected variants exhibit 3-oxo-C12 AHLaseimproved catalytic efficiencies compared to SsoPox wt. Theseimprovements range from 26 times for SsoPox W263T to 66 times for SsoPoxW263V with a k_(cat)/K_(M)=1.95(±0.44)×105 M⁻¹s⁻¹ that is the bestreferred to our knowledge (FIG. 3(C)). Concerning the phosphotriesteraseselected variants, none of them presents enhanced 3-oxo-C12 AHLasecatalytic efficiencies, only SsoPox W263F presents an efficiencyequivalent to the wt enzyme. SsoPox W263L, W263A and W263M lost theability to hydrolyse this molecule. The extent of the improvementsobserved makes of 3-oxo-C12 a potential promiscuous substrate.

Series of complementary results have been obtained for 3-oxo-C12 AHL,3-oxo-C10 AHL, δ-lactone and undecanoic-γ-lactone substrates (see table2′).

TABLE 2' Lactonase activities of W263 variants of SsoPox (ND correspondsto not determined value). SsoPox Enhancement/ variant k_(cat) (s⁻¹)K_(M) (μM) K_(I) (μM) k_(cat)/K_(M) (M⁻¹s⁻¹) wt 3-oxo-C12 wt 1.01 ± 0.13  456 ± 128 —  2.22(±0.68) × 10³ 1 AHL W263F 0.41 ± 0.02   146 ± 33 — 2.81(±0.65) × 10³  1.3 ± 0.5 (l) (XII) W263M ND ND — ND ND W263L ND ND— ND ND W263I 1.80 ± 0.05  17.8 ± 4.9 —  1.01(±0.28) × 10⁵  45.5 ± 18.8W263V 3.00 ± 0.07  24.7 ± 5.2 —  1.21(±0.26) × 10⁵  54.5 ± 20.4 W263T6.44 ± 0.22   137 ± 19 —  4.70(±0.67) × 10⁴  21.2 ± 7.2 3-oxo-C10 wt4.52 ± 0.10   143 ± 15 —  3.16(±0.40) × 10⁴ 1 AHL W263F 3.96 ± 0.18  288 ± 56 —  1.38(±0.28) × 10⁴ 4.4(±1.0) × 10⁻¹ (l) (XI) W263M ND ND —ND 0 W263L ND ND — ND 0 W263I (6.00 ± 0.90) × 10⁻¹  1605 ± 443 — 3.74(±1.17) × 10² 1.2(±0.4) × 10⁻² W263V (1.90 ± 0.09) × 10⁻¹  1346 ±298 —  1.41(±0.32) × 10² 4.5(±1.2) × 10⁻³ W263T (1.07 ± 0.16) × 10⁻¹ 1000 ± 343 —  1.06(±0.40) × 10² 3.4(±1.3) × 10⁻³ Undecanoic- wt 7.38 ±0.28   94 ± 18 —  7.86(±1.53) × 10⁴ 1 δ- W263F (6.65 ± 0.32) × 10¹ 135.2± 52.8 —  4.92(±1.93) × 10⁵  6.3 ± 2.7 lactone (r) W263M (7.12 ± 0.66) ×10¹   161 ± 47 7400 ± 2475  4.42(±1.35) × 10⁵  5.6 ± 2.0 (XX) W263L(5.68 ± 0.58) × 10¹   219 ± 62 4253 ± 1152  2.59(±0.78) × 10⁵  3.3 ± 1.2W263I (5.80 ± 0.74) × 10¹ <10  803 ± 213 >5.80(±0.74) × 10⁶ >73.8 ± 17.2W263V (4.48 ± 0.50) × 10¹    57 ± 16  789 ± 186  7.92(±2.34) × 10⁵  10.1± 3.6 W263T (9.33 ± 0.80) × 10¹   130 ± 41 3047 ± 576  7.17(±2.34) × 10⁵  9.1 ± 3.5 Undecanoic- wt 4.95 ± 0.26  2099 ± 230 —  2.36(±0.38) × 10³1 γ- W263F 4.63 ± 0.27   373 ± 111 —  1.24(±0.38) × 10⁴  5.3 ± 1.8lactone (r) W263M 4.25 ± 0.22   334 ± 61 —  1.27(±0.24) × 10⁴  5.4 ± 1.3(XVI) W263L 3.92 ± 0.17 371.8 ± 69.2 —  1.05(±0.20) × 10⁴  4.4 ± 1.1W263I 1.94 ± 0.08   361 ± 47 —  5.37(±0.73) × 10³  2.3 ± 0.5 W263V 5.64± 0.53  1760 ± 404 —  3.20(±0.80) × 10³  1.4 ± 0.4 W263T 4.55 ± 0.10 13.0 ± 4.2 —  3.49(±1.13) × 10⁵ 147.9 ± 53.5

Thermostability

The melting temperatures have been determined by circular dichroismspectroscopy for the wt SsoPox enzyme and the single position variants.Results are given below:

-   -   wt: 104° C.    -   W263F: 91.8±1.7° C.    -   W263M: 85.3±0.9° C.    -   W263L: 92.0±2.1° C.    -   W263T: 89.2±0.4° C.    -   W263V: 84.1±1.6° C.    -   W263I: 87.8±1.2° C.

2.2.2—Multiple Positions Variants

Some of the above mentioned mutated hyperthermophilic phosphotriesterase(PTE) having a lactonase activity derived from a hyperthermophilicphosphotriesterase corresponding to the sequence of SEQ ID NO: 3 havebeen tested for their ability to hydrolyse either OPs or AHLs compounds.The results of their enzymatic activities are presented hereafter.

Five mutated hyperthermophilic phosphotriesterase (PTE) having alactonase activity derived from the hyperthermophilic PTE of Sulfolobussolfataricus corresponding to the sequence SEQ ID NO: 3 have been testedfor their phosphotriesterase activity. The evaluation ofphosphotriesterase activity has been performed using ethyl-paraoxon,methyl-paraoxon, ethyl-parathion, methyl-parathion and malathion.Results are presented in tables 4 and 5 hereafter.

TABLE 4 Phosphotriesterase activity of variants of SsoPox αsA1, αsA6,αsB5. Catalytic avtivity is expressed in M⁻¹s⁻¹ (ND = not detected, VLH= very low hydrolysis). SsoPox αsA1 αsA6 αsB5 Substrat wt SEQ ID NO: 21SEQ ID NO: 27 SEQ ID NO: 29 Ethyl-Paraoxon 5.19 (±1.31) · 10² 3.37(±0.94) · 10⁴ 3.61 (±1.69) · 10³ 4.98 (±0.94) · 10⁴ Methyl-Paraoxon 1.27 (±0.7) · 10³ 2.29 (±1.09) · 10⁴ 1.08 (±0.30) · 10⁴ 4.31 (±0.14) ·10³ Ethyl-Parathion ND VLH 2.39 (±0.47) · 10² 9.32 (±1.44) · 10²Methyl-Parathion 9.09 ± 0.9  3.68 (±0.5) · 10¹ 61 ± 15 9.49 (±1.15) ·10² Malathion 5.56 ± 1.26 3.2 ± 0.7 ND 31.1 ± 7.7

TABLE 5 Phosphotriesterase activity of variants of SsoPox αsC6 and αsD6.Catalytic avtivity is expressed in M⁻¹s⁻¹ (ND = not detected, VLH = verylow hydrolysis). SsoPox αsC6 αsD6 Substrat wt SEQ ID NO: 31 SEQ ID NO:23 Ethyl-Paraoxon 5.19 (±1.31) · 10² 2.86 (±0.17) · 10⁴ 6.22 (±1.01) ·10⁴ Methyl-  1.27 (±0.7) · 10³ 3.11 (±1.32) · 10⁴ 2.04 (±0.59) · 10⁴Paraoxon Ethyl-Parathion ND 1.10 (±0.20) · 10² 6.05 (±1.50) · 10³Methyl- 9.09 ± 0.9  24.8 ± 3.9 2.01 (±0.36) · 10⁴ Parathion Malathion5.56 ± 1.26  7.7 (±5.94) · 10² 4.20 (±0.49) · 10²

Among the phosphotriesterase selected variants, SsoPox asD6 exhibits thehighest paraoxonase catalytic efficiency for ethyl-paraoxon,ethyl-parathion and methyl parathion. SsoPox αsC6 exhibits the highestparaoxonase catalytic efficiency for malathion. Unlike SsoPox wt, SsoPoxαsA6, αsB5, αsC6 and αsD6 are now able to hydrolyze methyl parathion.

SsoPox αsD6 is probably the most interesting variant of SsoPox for itscapacity to hydrolyze several OPs subtrats.

Example 2

In this example, we tested whether the variant SsoPox W263I, which hasan improved ability to hydrolyze 3-oxo-C12 AHLs could decrease P.aeruginosa biofilm formation and virulence factor production in vitro,and reduce mortality in vivo. We present evidence thatlactonase-mediated quorum quenching inhibits virulence and decreaseslethality of P. aeruginosa in a rat pulmonary infection model.

1—Experimental Procedure

1.1—P. aeruginosa Culture

The laboratory strain PAO1 (ATCC reference 15692) was used in allexperiments. Strains were grown in LB (BD, France) medium and weremaintained at −80° C. in 50% LB broth and 50% glycerol. P. aeruginosaPAO1 carrying a chromosomally integrated PlasB-luxCDABE reporterconstruct [11] was maintained in the same way as the wild-type strain.Strains were grown at 37° C. in Luria-Bertani (LB) medium (BD, France)with shaking (200 rpm). LB was solidified with 1.5% bacto agar whenrequired.

For in vivo studies, at the time of the experiments, aliquots containingthe bacteria were thawed and cultured on a COS (Biomerieux, France)(Columbia with 5% Sheep blood) agar plate. Ten fresh colonies weresampled and cultured overnight at 37° C. in triptych soy broth (TSB,Biomerieux, France) with continuous agitating until the OD_(600 nm)=1with cultured PAO1. Serial dilution was subsequently performed to adjustthe bacterial amount and exact concentrations were confirmed by platingserial dilutions on the appropriate culture medium and countingcolonies. Inoculums of 10⁸ CFU/ml were used for all animal infections.

1.2—Biofilm Formation Assays

Cultures of P. aeruginosa PAO1 (18 hours) were diluted 1:50 in 10% TSBand dispensed into the wells of a Calgary Biofilm Device 96 well plate(Innovotech Inc., Edmonton, Canada). To test inhibition of biofilmformation, three-fold dilution series (50 μg down to 0.5 μg of SsoPoxW263I) was added to the wells. Plates were incubated for 4 hours withrocking at 120 Hz at 37° C. The biofilms were stained with 1% crystalviolet. Crystal violet was dissolved from biofilms in 100% ethanol andquantified by measuring absorbance at 600 nm [14]. P. aeruginosa PAO1planktonic growth was also measured at 600 nm.

1.3—LasB Reporter System

Aliquots of P. aeruginosa PAO1 carrying PlasB-luxCDABE from an 18 hr oldculture were placed in wells of a 96 well plate, after which dilutionsfrom 50 μg to 0.05 μg of SsoPox W263I were added. Plates were incubatedat 37° C. for 90 minutes, with shaking every 10 minutes during whichluminescence was measured every 10 minutes to determine activity of thequorum sensing reporter.

1.4—Animal General Procedure

Adult Sprague-Dawley male pathogen-free rats weighting 250 to 300 g fromSAS Janvier (Le-Genest-St-Isle, France) were housed in individualplastic cages placed in a ventilated pressurized cabinet (A-BOX 160,Noroit, Reze, France) with free access to water and standard diet food.Animals were anesthetized with 5% Sévoflurane® (Abbott, Rungis, France)in 100% oxygen (anesthetizing box, Harvard Apparatus, Les Ulis, France).Their trachea was exposed and intubated using a 16-gauge catheter fordrug and/or bacterial administration. Awaken animals were housed back inthe same condition as initially and were weighed daily. At the end ofeach experiment, euthanasia was performed with an intra-peritonealinjection of a lethal dose of thiopental (Panpharma, France).

1.5—Rat Tolerance of Inhaled SsoPox W263I

The tolerance of SsoPox administered by intra-tracheal route wasattested in a preliminary study on 3 groups of animals (n=3 per group)receiving 250 μl of SsoPox W263I at a concentration of either 0.1, 1 or10 mg/ml and compared to 5 controls receiving 250 μl of phosphatebuffered saline (PBS; Biomerieux; France). One animal of each group wassacrificed after 6, 24 and 48 hours. Surviving animals were sacrificedafter 48 hours. Lungs were removed after death and their macroscopicaspect was noted, then they were preserved in formaldehyde forhistological assessment.

1.6—Rat Respiratory Infection Model and SsoPox W263I Treatment.

Three groups of 20 animals were infected by intra tracheal inoculationof 250 μl of a solution of PBS containing 10⁸ CFU/ml of P. aeruginosaPAO1. At the same time, a first group received 250 additional l of PBSinto the trachea (non-treated group: NT), another group received 250 μlof SsoPox W263I at a concentration of 1 mg/ml (immediate treatmentgroup: IT). In the last group, animals received 250 μl of SsoPox W263Iat 1 mg/ml 3 hours later (deferred treatment group: DT). SsoPox W263Iand additional PBS were delivered intratracheally using the sameanesthetic procedure as for the infection.

1.7—Lung Processing

After infection, animals were observed for 2 days and spontaneousmortality was noted. Surviving rats were euthanized after 48 hours.After death, lungs were removed aseptically. Right lung was homogenizedin PBS for bacterial culture. Left lung was preserved for histologicalanalysis.

1.8—Histological Severity Score (HSS)

Examination was performed by a pathologist blinded to the group identity(H. L.). An HSS was calculated based on the number of bronchopneumonialesions (0, no lesions; 1, 30 lesions/lung; 2, ≥30 lesions/lung; 3,confluent lesions of bronchopneumonia), as previously reported[13].

1.9—Statistics

The number of studied animals (20 animals per group) was calculatedbased on a mortality reduction from 80% in the NT group infected withPAO1 (known from literature data [12]) to an expected mortality rate of50% in the treated groups, with 90% statistical power and a two-sidedalpha value of 0.05.

2—Results

2.1—Decreases of lasB Expression and Biofilm Formation by SsoPox W263I

We measured lasB transcription in a P. aeruginosa PAO1 strain carryingthe chromosomally integrated PlasB-luxCDABE reporter construct. The genelasB codes for elastase, a classical virulence factor regulated byquorum sensing. Addition of SsoPox W263I into P. aeruginosa PAO1cultures significantly reduced lasB transcription (FIG. 4). SsoPoxW263I-mediated lasB inhibition exhibited a dose-dependent profile with ahalf inhibition concentration ([C_(1/2)]) of the enzyme around 0.5 μg/mL(FIG. 4).

Biofilm development is also regulated in part by quorum sensing. Theeffect of SsoPox W263I on the ability of P. aeruginosa to form biofilmswas investigated. Our results show that the lactonase inhibits biofilmformation in a dose-dependent manner, with a [C_(1/2)] of approximately170 ag/mL (FIG. 5).

2.2—SsoPox W263I Protects Rats from P. aeruginosa PAO1 Pneumonia

The effects of SsoPox treatment on rat respiratory tissues wereinvestigated.

Tracheal instillation of SsoPox W263I was well tolerated—no spontaneousmortality was observed regardless of the dose administered (up to 2.5mg). Lungs removed after treatment showed no macroscopic signs of injuryand histological analysis showed normal lung parenchyma. SsoPox W263Icaused no observable acute inflammatory reactions in the rat respiratoryparenchyma.

The influence of SsoPox W263I on P. aeruginosa pulmonary infection wasinvestigated in two groups of 20 rats. In the non-treated group (NT),the mortality rate after inoculation with P. aeruginosa was 75% (15/20).When the rats were treated with SsoPox (1 mg/mL) immediately afterinoculation with P. aeruginosa (IT), the mortality was significantlyreduced to 20% (4/20) (p=0.0001 vs NT) (FIG. 6). In addition we observedthat loss of body weight, measured from the day of inoculation with P.aeruginosa until the day of death, was significantly less in the ITgroup than in the NT group (11.3±12 g vs. 20.4±9.3 g respectively;p=0.01). The DT group lost 25.6±1.82 g of body weight (p=0.77 vs NTgroup). Moreover, mean time to death was significantly longer in the DTgroup as compared to the control group (37±13 vs. 25±16 hours; p=0.01).

Consistent with the increased survival of the IT group, we noted thatthe lungs of the animals in the IT group had less inflammatory damage ascompared to the NT group (FIG. 7): HSS (1.27±0.6 vs. 2.64±0.4; p=0.005).In the DT group, the mean HSS was not different from the NT group.

Example 3

The ecotoxicity of SsoPox has been tested on the viability anddevelopment of oyster larvae (Crassostrea gigas) and sea urchins larvae(Paracentrotus lividus) during 24 hours and 48 hours respectively.Experiments have been done using 10 mg/l, 1 mg/l, 100 μg/l, μg/l, 1 μg/lor 100 ng/l of SsoPox and two samples of at least 100 larvae have beenanalyzed. CuSO4 has been used as a toxic control.

In the case of the urchin larvae, no effects have been observed at anyof the tested concentrations.

In the case of the oyster larvae, no effects have been observed for aconcentration equal or lower to 1 mg/L, only 10% of the population isaffected at a concentration of 2.9 mg/L (sample 1) or 3.5 mg/L (sample2).

These results indicate that high concentrations of SsoPox are not toxicfor living organisms and thus, the use of SsoPox in sea environment canbe considered favorably.

REFERENCES

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1. An antibacterial composition comprising as active ingredient at leastone mutated hyperthermophilic phosphotriesterase, said mutatedhyperthermophilic phosphotrieterase has an increased lactonase catalyticactivity in comparison of the lactonase activity of a non-mutatedhyperthermophilic phosphotriesterase, wherein the non-mutatedhyperthermophilic phosphotriesterase is a wild-type phosphotriesterasecorresponding to the consensus sequence of SEQ ID NO:1, wherein theamino acid W in position 265 is substituted by an amino acid selectedfrom the group consisting of isoleucine I, valine V, threonine T andalanine A within the mutated hyperthermophilic phosphotriesterase. 2.The antibacterial composition according to claim 1, wherein hydrolysisof 3-oxo-C12 AHL by said mutated hyperthermophilic phosphotriesterase isincreased by at least 2 times, in comparison to hydrolysis of 3-oxo-C12AHL by said non-mutated hyperthermophilic phosphotriesterase.
 3. Theantibacterial composition according to claim 1, wherein said mutatedhyperthermophilic phosphotriesterase has a thermostability, which issubstantially similar to the thermostability of said non-mutatedhyperthermophilic phosphotriesterase.
 4. The antibacterial compositionaccording to claim 1, wherein the amino acid in position 2 in SEQ ID NO:1 of said non-mutated hyperthermophilic phosphotriesterase is missing.5. The antibacterial composition according to claim 1, wherein saidnon-mutated hyperthermophilic phosphotriesterase is selected from thegroup consisting of SEQ ID NO: 3 from Sulfolobus solfataricus, SEQ IDNO: 5 from Sulfolobus acidocalaricus, and from SEQ ID NO: 7 Sulfolobusislandicus, wherein said sequences SEQ ID NO: 3, SEQ ID NO: 5 and SEQ IDNO: 7 belong to the consensus SEQ ID NO: 1, and for said mutatedhyperthermophilic phosphotriesterase the amino acid in position 2 in SEQID NO: 1 being missing from SEQ ID NO: 5 and the amino acids in position2 and 3 in SEQ ID NO: 1 being missing from SEQ ID NO: 3 and SEQ ID NO:7.
 6. The antibacterial composition according to claim 1, wherein saidamino acid W in position 265 is substituted by an amino acid IsoleucineI within said mutated hyperthermophilic phosphotriesterase.
 7. Theantibacterial composition according to claim 1, said mutatedhyperthermophilic PTE being chosen in the group consisting of: SEQ IDNO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 57, SEQID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 101, SEQ ID NO: 103,SEQ ID NO: 105 and SEQ ID NO:
 107. 8. A phytosanitary compositioncomprising the antibacterial composition according to claim
 1. 9. Apharmaceutical composition comprising the antibacterial compositionaccording to claim 1, said antibacterial composition further comprisinga pharmaceutically acceptable vehicle.
 10. The pharmaceuticalcomposition according to claim 9, further comprising at least oneantibiotic selected from the group consisting of gentamycine,ciprofloxacin, ceftazidime, imipenem, and tobramycine.
 11. A method oftreating a bacterial infection, comprising administering to a patient inneed thereof the antibacterial composition according to claim
 1. 12. Themethod of treating bacterial infections according to claim 11, whereinthe bacterial infection is pneumonia or nosocomial diseases, caused bybacteria using homoserin lactone substrates to communicate, inparticular in the blood, wounds, burn, skin, biomaterial-body contactarea.
 13. A method of disrupting quorum-sensing in bacteria comprisingadministering the antibacterial composition according to claim
 1. 14.The method according to claim 13, wherein the antibacterial compositionis administered to boats or other sea equipment and limits the formationof biofilms in boats or other sea equipment.
 15. The method according toclaim 13, wherein the antibacterial composition is administered toplants or vegetables and inhibits fire blight in plants or rotting ofvegetables.